FI92714C - Apolipoprotein B-specific monoclonal antibodies produced by two novel hybridomas - Google Patents

Abstract

Two hybridomas that produce receptors containing antibody combining sites that immunoreact with apolipoprotein B-100 are disclosed as are uses for the receptors, compositions and diagnostic systems that include the receptors.

Description

! 92714

Apolipoprotein B-specific monoclonal antibodies produced by two new hybridomas

The present invention relates generally to novel hybridomas, and more particularly to hybridomas that produce receptor molecules that immunoreact with apolipoprotein B-100, receptor molecules so produced, as well as diagnostic methods and systems using receptor molecules and.

Lipoproteins are the primary transporters of plasma cholesterol and triglycerides. They are micellar lipid-protein complexes that contain a protein (called an apoprotein) and polar lipids arranged in a surface membrane that surrounds a neutral lipid core (triglyceride and cholesteryl ester). Lipoproteins were initially identified based on their specific weights as measured by ultracentrifugation. Accordingly, four classes of bulk densities are known: chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL).

In parallel with advances in ultracentrifugal separation technology, the LDL and HDL density classes have been further subdivided into four subclasses with greater homogeneity. For example, LDL can be divided into medium density lipoprotein (IDL) and LDL2 subclasses. However, the Namak subclasses consist of functionally heterogeneous populations of lipoprotein particles due to their varying apoprotein content.

Eight major proteins, A1, A-II, A-IV, B, CI, C-II, C-III, and E, have been isolated, and from the group of waxy aoproteins that can be recovered in higher amounts from certain density classes, most can also be found in other density classes. Thus, most LDL particles contain only 2,971 4 of apo B, however, some particles also contain other apoproteins, and this explains their small proportions of apo CI, apo C-II, C-III, and apo E. which are lasna in this density class.

In some cases, certain functions have been considered to belong to certain apoproteins. For example, an apo B species synthesized in the liver, called apo B-100, recognizes and binds to cellular LDL receptors. By binding apo B-100, these receptors bind LDL particles and withdraw them from the plasma. LDL enters the cells and is broken down, releasing its cholesterol according to the needs of each cell. The Apo B-LDL receptor interaction thus plays a significant role in the removal of LDL cholesterol from the blood.

The LDL receptor does not recognize another apo B species called apo B-48. Tata apo B species, which is only 48% of the total apo B-100, is synthesized in humans only in the gut. Liooproteins containing apo B-48, such as chylomicrons and chylomicron ducts, do not bind to the LDL resorotor.

Although these two apo B species appear to be genetically isolated (a single patient has developed apo B-48 but not aoo B-100), immunological studies have shown that apoproteins B -100 and B-48 are contiguous antigenic sites (determinants). At least three research teams have reported the development of a total of seven different monoclonal antibodies that bind to either apo B-100 or apo B-48. From the results reported by those researchers, it can be clearly concluded that apo B-48 and apo B-100 are similar in structure; i. that apo B-48 may correspond to part of the apo B-100 protein. There is also evidence that apo B-48 and apo B-100 are not found in the same lipoprotein particle, indicating that separate apo B particles exist.

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Several researchers have recently suggested that plasma apo B levels may provide a better indication of coronary artery disease (CAD) risk than plasma LDL cholesterol levels. Sniderman et al., Proc. Natl. Acad. Sci. USA 77, 604-608 (1980). As atherosclerosis and related complications continue to be the leading cause of death and weakness in the West, the medical industry has long felt the need to find configuration systems capable of identifying those at risk for CAD.

Many types of plasma apoprotein B immunoassays have been reported utilizing antisera containing specific antibiotics, including competitive liquid and solid phase radioimmunoassays (RIAs), enzyme-linked immunosorbent assays, ELISAs, ELISAs. Problems that limit the widespread use of these Apo B immunoassays by Levin have been reproducibility and the quality and specificity of the anti-sera used. Compilation articles on methodological problems for each different type of apo B assay have been written by Currey et al., Clin. Chem. 24, 280-286 (1978) and Rosseneu et al., Clin. Chem. 28, 427-433 (1983).

Several researchers have reported the development of panels containing monoclonal antibodies against human apo B for use in studying its antigenic structure and lipo-protein metabolic significance. In addition, there have been reports of the use of anti-apo B monoclonal antibodies to measure plasma apo B levels in liquid phase RIA assays. Patton et al., Clin. Chem. 29, 1898-1903 (1983) and Maynard et al., Clin. Chem. 30, 1620-1624 (1984). In addition, one group has reported the use of a mixture of anti-apo B monoclonal antibodies in a plasma apo B radial immunodiffusion assay. Marconvina et al. Clin. Chim. Acta 147, 117-125 (1985). However, these assay methods are hampered by the need for long incubations, repeated 4,92714 centrifugations, or the use of radioactive materials.

The use of monoclonal antibodies as reagents in determining the presence of apo B-100 in human body fluid samples is attractive because such reagents can be produced in relatively large amounts uniformly after preparation. However, there are a number of factors that oppose the use of a particular monoclonal antibody as a component in the apo B-100 detection system.

First, it is known in the art that a monoclonal antibody may be too immunospecific to be useful due to the antigenic heterogeneity of the target antigen. The specificity of conventional antisera containing polyclonal antibodies depends on the parallelism of hundreds of thousands of different antibodies that bind to antigenic determinants and cover most or all of the antigenic protein. As a result, small changes in the structure of the antigen, due to genetic diversity, heterogeneity in glycosylation, or mild denaturation, usually contribute well to the binding of a waxy polyclonal antibody. Similarly, a variable subset of antibodies from polyclonal antisera bind antigens that have been modified or denatured.

In contrast, monoclonal antibodies usually bind to a single antigenic determinant (epitope) on an antigenic molecule. If, for one reason or another, that detergent is altered, the antibiotic may or may not continue to bind. Whether this is a problem or an advantage depends on individual circumstances. If, as in the present case, the monoclonal antibody is to be used in the diagnostic determination of an apoprotein, a waxy antigenic modification in that protein could cause obvious errors.

The antigenic heterogeneity of apoprotein B-100 is well documented. For example, the expression of an apo B epitope is

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5,927 4 have been found to vary depending on (1) the composition of the associated lipids, (2) the temperature state of the immunoreaction, (3) the degree of isolation of LDL from its natural environment, and (4) the genetic expression between individuals.

Second, due to its unique specificity, the successful use of a monoclonal antibody (MoAb) often depends on its affinity for the target antigen. For example, while the affinity of MoAb may be sufficient to be a Hyiod when binding a liquid and solid phase antigen, while the MoAb itself is in the liquid phase, the same antibody may not be useful as a solid-state antibody useful for antigen binding and "withdrawal". .

The above problems are characteristic of the use of monoclonal antibodies. Those skilled in the art have therefore found that it is essential to test and characterize monoclonal antibodies in each assay system in which they are to be used. See Goding, James W. (Monoclonal Antibodies: “Principles and Practice.” Pages 40-46, Academic

Press, New York (1983).

One aspect of the present invention contemplates a hybridoma designated HL130C2.3C5 having ATCC Accession No. HB 8746. The hybridoma and its receptor molecules are also referred to herein as MB47.

In another aspect, the present invention contemplates receptor molecules that immunoreact with aoprotein B-100 and are secreted by the hybridoma ATCC HB 8746.

Yet another aspect of the present invention contemplates a hybridoma designated V82A6.1G4 having an ATCC Accession No. HB 8742. The hybridoma and its receptor molecules are also referred to herein as MB24.

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Yet another aspect of the present invention contemplates receptor molecules that immunoreact with apoprotein B-100 and are secreted by the hybridoma ATCC HB 8742.

Another aspect of the invention in accordance with the present invention is a cell culture comprising (a) a hybridoma of the present invention; (b) receptor molecules secreted by said hybridoma that immunoreact with apoprotein B-100; and (c) culture medium for hybridoma.

According to a further aspect of the invention, there is provided a method of determining the presence of apoprotein B-100 in a body fluid sample, comprising the steps of: (a) taking a body fluid sample to be determined; (b) producing receptor molecules in a biologically active form that (i) immunoreact with apoprotein B-100 and (ii) secreted by either hybridoma HB 8746 or HB 8742; (c) mixing the body fluid sample with the receptor molecules: (d) maintaining the mixture under biological assay conditions for a predetermined time sufficient for the receptor molecules to immunologically bind the apoprotein B-100 in the sample to form an immunoreactant; and (e) determining the amount of immunoreactant formed.

In another aspect, in accordance with the present invention, there is provided a method of determining apoprotein B-100 from a body fluid sample, the method comprising the steps of: (a) taking a body fluid sample to be determined; (b) providing a solid support that encapsulates a solid matrix to which is attached a first receptor in a biologically active form that immunoreacts with apoprotein B-100 and is secreted by a hybridoma having ATCC Accession No. HB 8746; (c) producing a biologically active second receptor which immunoreacts with apoprotein B-100 and secreted by a hybridoma having ATCC Accession No. HB 8742, said second receptor being associated with a labeled enzyme capable of expressing the presence of said second receptor in the immunoreactant; (d) mixing substantially simultaneously: (i) said body sample; (ii) a first receptor; and (iii) a labeled second receptor to form a solid / liquid phase immunoreaction mixture; (e) maintaining the mixture under biological assay conditions for a predetermined time sufficient for the first receptor and the labeled second receptor to immunologically bind the plasma apoprotein B-100 to form a solid phase, layered immunoreactant; (f) separating said solid phase, layered immuno-reactant from the liquid phase; and (g) determining the amount of labeled second receptor bound in the resulting solid phase, layered immunoreactant.

From another aspect of the present invention, there is provided a competitive method for determining apoprotein B-100 in a body fluid sample, the method comprising the steps of: (a) taking a body fluid sample to be determined; (b) providing a solid support that encapsulates a solid matrix having a predetermined amount of the apoprotein B-100 reagent attached; (c) substantially simultaneously mixing (i) a sample of the body; (ii) a solid carrier; and (iii) a predetermined number of receptor molecules that immunoreact with apoprotein B-100 secreted from either a hybridoma having an ATCC accession number of HB 8746 or a hybridoma having an ATCC accession number of HB 8742. to form a liquid mixture; 9271 4 8 (d) maintaining the mixture under biological assay conditions for a time sufficient for the receptor molecules to bind immunologically to the apoprotein B-100 molecules on a solid support and to the apoprotein B-100 molecules present in the body fluid sample; (e) separating the solid phase immunoreactant from said liquid phase; (f) mixing the solid phase immunoreactant with a biologically active second receptor that immunoreacts with the first receptor to form a second solid / liquid phase immunoreaction mixture, the second receptor being associated with a labeled enzyme capable of detecting the presence of said second receptor as an immunogen; (g) maintaining said second mixture under biological assay conditions for a time sufficient for the labeled second receptor to immunologically bind to the first receptor, which is a solid phase immunoreactant, to form a solid phase layered immunoreactant; (h) separating the solid phase, layered immunoreactant from the liquid phase; and (i) determining the amount of labeled second receptor bound to said solid phase, layered immunoreactant.

In another aspect, the present invention provides a cell culture comprising: (a) a hybridoma having ATCC Accession No. HB 8746; (b) receptor molecules secreted by the hybridoma that immunoreact with apoprotein B-100; and (c) culture medium for hybridoma.

In another aspect, according to the present invention, there is provided a composition comprising: (a) a hybridoma having ATCC Accession No. HB 8742;

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92714 9 (b) hybridoma-secreted receptor molecules that immunologically react with apoprotein B-100; and (c) culture medium for hybridoma.

In another aspect, in accordance with the present invention, a diagnostic system for determining the amount of apo B-100 in a body sample comprising: (a) a biologically active first specific binding agent that encodes receptor molecules (s) that encode receptor molecules; with; and (ii) are either receptor molecules secreted by the hybridoma ATCC HB 8746 or receptor molecules secreted by the hybridoma ATCC HB 8742; and (b) a biologically active, labeled second specific binding agent to detect immunoreaction of the first binding agent with apo B-100.

A solid matrix affinity sorbent that encapsulates a solid-phase matrix attached to biologically active receptor molecules that immunoreact with apoprotein B-100 and is selected from the group consisting of: (a) receptor molecules secreted by hybridoma HB 8746; (b) receptor molecules secreted by the hybridoma ATCC HB 8742; and (c) a mixture comprising the receptor molecules of (a) and (b).

The present invention provides several benefits and advantages.

The advantage of the present invention is that the hybridoma of the present invention can be used to produce relatively large amounts of homogeneous receptors that immunoreact with apo B-100.

Another advantage of the present invention is that the receptors of the present invention are useful, inter alia, in determining the amount of cholesterol-carrying apo B-100 in a body fluid sample.

The advantage of the present invention is that the receptors of the present invention can be used in enzyme-linked immunosorbent assays of apo B-100 in formats that do not require centrifugation.

Another advantage is that the assay methods of this invention can be completed in relatively short periods of time.

Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following description, drawings, and appended claims.

Brief description of the drawings

Figure 1 shows a photograph of a Western blot autoradiogram showing the ability of MB47 and MB24 receptor molecules to immunoreact with apo B-100 and apo B-48 obtained from lipid-free chylomicrons and VLDL. VLDL and chylomicrons were delipidated and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 3-6% gradient gels. Sixty micrograms (μg) of VLDL protein and 20 μg of chylomicron protein were run in alternating bands on the gel, thus separating the proteins of each preparation by size. Visualization of an electrophoresis strip containing protein bands, masking the gel with 0.1% Coomassie Blue vari, revealed apo B-100 and apo B-48 bands in chylomicrons and VLDL. The protein-containing bands were then attached by electrophoretic transfer to nitrocellulose paper to provide a solid support. The apoprotein antigens attached to the solid support were then separately immunoreacted with immunopurified MB47 and MB24 receptor molecules. Monoclonal MB47 and MB24 monoclonal receptor molecules immunologically bound to 9271 4 11 solid phase antigen were expressed using 125 ...

this J-labeled goat anti-huri Ig 3a autoradiography.

Panel A depicts that monoclonal MB24 immunoreacts with apo B-100 and apo B-48 from VLDL (V) and chylomicrons (C). Panel B depicts that monoclonal MB47 immunoreacts with apo B-100 from V and C, but not with apo B-48 from either V or C. Panel C is a negative control showing that a monoclonal antibody specific for sheep erythrocytes does not immunoreact with any antigen in the sheep. Panel D is another negative control showing that the immunopurified polyclonal antiserum to phenyl-beta-O-glucoside does not recognize any lasal antigen. Panel E is a positive control showing that immunopurified rabbit polyclonal antiserum against human LDL apoprotein recognizes both apo B-100 and apo B-48 in both V and C.

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Figure 2 is a graph showing the percentage (ordinate) of bound J-labeled LDL particles with increasing molar concentration / abscissa of monoclonal antibody MB47; Ab concentration (MoAb _) _ / liquid phase radioimmunoassay (RIA).

LDL was prepared from pooled plasma (----) or one normal fat-blooded person (_).

Plasma was obtained by plasma aphasia of individuals after a fasting period of approximately twelve hours.

Figure 3 is a bar graph showing the degree of inhibition of antibody MB47 (empty bars: MB47) and excess human unlabeled LDL (dashed bars: LDL) to 125 human J-LDL binding, entry, and degradation by cultured human fibroblasts. Unicellular layers of fibroblasts were cultured in 35 mm wells in DME containing 10% fetal calf serum.

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Fibroblast LDL receptors were stimulated by approximately 24 hours of preincubation of fibroblasts in medium containing 2.5 milligrams per milliliter (mg / ml) of lipoprotein-free serum (LDS) (DME-LDS). DME-LDS containing 12 5 2.5 micrograms per milliliter / ml) of J-LDL and either 20% MB47 hybridoma culture supernatant (v / v) or 200-fold excess of unlabeled LDL (final concentration). thio, 500 μg / ml), were mixed and kept (incubated) for about 16 hours at 4 ° C before addition to the fibroblast monolayers.

Binding, uptake, and degradation assays were performed in triplicate and are expressed as a percentage of control values determined in the absence of monoclonal receptor MB47. The inhibition of specific binding, transport, and degradation by the monoclonal receptor MB47 was comparable to that produced by a 200-fold excess of unlabeled LDL.

Figure 4 contains two curves showing the ability of monovalent Fab fragments of monoclonal antibody MB47 to inhibit the binding and degradation of 125 I-human J-LDL by human fibroblasts. Individual media containing growing 12 5 MB MB47 Fabs and standard J-LDL (2.5 μg / ml) were mixed and kept (incubated) for about 15 hours at 4 ° C before addition. fibroblast monolayers. The Fab concentration is expressed as the molar Fab / LDL ratio, which is the plasma in each medium (assuming a molecular weight (MW) of Fab of 40,000 daltons and a molecular weight of apo B of 550,000 daltons).

Binding and degradation are expressed as a percentage of control values in the absence of MB47 Fab fragments. All determinations were performed in parallel. Excess unlabeled LDL (final concentration, 500 ug / ml) produced more than 95% inhibition of J-LDL binding (panel A) and degradation.

* t 13 92714 (panel B). Similar results were obtained in three studies with different Fab preparations of the monoclonal receptor MB47.

Figure 5 contains two graphical representations. Figure A depicts the ability of pepper root peroxidase-labeled MB47 (HRPO-MB47) receptors, which are known to be constant, to immunoreact with solid phase reagent apo B-100 in the presence of increasing numbers of MB24 receptor molecules. The ordinate is in units of relative optical density, while the abscissa is in micrograms / milliliter of unlabeled antibody proteins added (μg / ml) as a competitor.

A constant amount (20 μg) of MB47 receptors coupled to HRPOr was mixed substantially simultaneously with growing amounts of unlabeled MB47 (·) or unlabeled MB24 (A) receptors and solid phase reagent apo B-100 (LDL). The mixtures were kept for 3 hours at 25 ° C, thus allowing the receptors to immunologically bind the reagent apo B-100 and form a solid phase immunoreactant. The amount of labeled MB47 bound to the solid phase was then determined as in the competitive ELISA described in the Materials and Methods section.

Graph A depicts that as the amounts of unlabeled MB47 receptors increase in the immunoreaction mixture, the number of labeled MB47 receptors bound as a solid phase immunoreactant decreases accordingly. The unlabeled MB47 thus competes with the labeled MB47 for LDL.

Graph A, on the other hand, also reflects the fact that as the number of unlabeled MB24 receptors increases, the amount of labeled MB47, which is bound as a solid phase immunoreactant, does not decrease significantly. Thus, unlabeled MB24 did not compete with labeled MB47 for binding to LDL.

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Graph B illustrates that similar results are obtained using HRPO-labeled MB24 receptors and unlabeled MB47 receptors. The MB47 and MB24 receptors therefore bind to different epitopes that are sufficiently separated on the surface of apo B-100 to allow binding of both receptors to a single apo B-100 molecule without steric competition and inhibition of binding.

Figure 6 contains two graphical representations. In Presentation A, Brochure 12 describes the binding of J-labeled B-47 to LDL in a liquid phase RIA. The ordinate is shown in units of femtomole (fmole) of bound, while the abscissa is in units of nanomolar (nM) of antibody mixed.

Immunologically purified monoclonal antibody MB47 125. .

iodinated with J using Iodogen technique to a specific activity of 3000 pulses per minute per nanogram (cpm / ng). After thorough dialysis against phosphate buffered saline (PBS), more than 95% of the radioactivity was precipitated with 10% trichloroacetic acid (TCA). More than 98% of 125 I-MB47 bound to the LDL column. The determinations were performed in triplicate in 10 x 75 mm-125 meter (mm) silicone bale glass tubes. J-MB47 was added in increasing concentrations in 0.1 ml of bovine serum albumin-barbital (BSA-barbital) buffer (pH 8.0) to 100 ng of pooled, normal lipid-diluted human LDL diluted 0.2 per ml of BSA-barbital buffer. Each tube contained 182 fmoles of LDL apo B (assuming a molecular weight of apo B of 550,000 daltons).

After 16 hours of incubation (mixing and standing) at 4 ° C, LDL was quantitatively precipitated with lipoprotein-free rabbit antiserum specific for human LDL. / Only the fraction of rabbit antiserum with a density (d) greater than 1.21 g / ml was used because monoclonal antibody MB47 binds rabbit apolipoprotein Brta ^ / 9271 4 15

Preliminary studies showed a concentration of rabbit delipidated antiserum that precipitated more than 98% of 100 ng of 125 human J-LDL. After addition of rabbit antiserum, the tubes were incubated for about 16 hours at 4 ° C.

The supernatants were removed and the pellets were washed twice with 2 ml of cold barbital buffer (pH 8.0). Nonspecific binding and precipitation were determined in two parallel tube batches. In the first batch, no human LDL was added to the initial incubation, but the same amount of rabbit second antibody was added. In another group of tubes, rabbit non-immune serum (fraction with d greater than 1.21 mg / ml) replaced rabbit immune serum antibody.

Both methods yielded substantially similar values and non-specific binding, which was linear with increasing concentrations of 125 I-MB47 monoclonal antibody and was less than 1% of the total total added in all tapas. Specific binding of J-MB47 to LDL was obtained by reducing non-specific binding from total binding. Binding results were analyzed using the linear regression program of the Scatchard assay used in ligand binding systems, which gave an estimate of the antibody affinity constant (Ka) and receptor or epitope concentration.

The Ka for LDL of J-MB47 was thus determined to be 3.82 x 10 ^ M Extrapolation of the line to correspond to the conditions in the treasure-free excess of antibody gave an estimate of 182 fmoles (100 ng) of LDL bound to 212 fmoles (35 ng) ^ ^ J-MB47, when the molecular weight of apo B is assumed to be 550,000 daltons. These results are shown in Graph B of this figure, where the ordinate is shown as the ratio of bound (B) to free (F) antibody (B / F), while the abscissa is shown as the molar units of bound antibody (fmoles of MB47). .

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Figure 7 depicts the correlation between mg of LDL cholesterol per deciliter (dl) as determined by the method of Lipid Research Clinic Procedures, HEW Publication No. 75-628 (NIH), 2nd Edition, Wash., D.C. Goc. Print Off. (1974), and mg of apo B-100 as determined by navel dehydration using the non-competitive ELISA method described in the Materials and Methods section.

Correlation coefficient, r = 0.89, determined by the Spearman Rank Correlation test, parameter-free results vårten / Sokal et al., Biometry, 2nd edition, W.H. Freeman Co., San Francisco, CA, 561-616 (19812), shows a significant correlation between apo B-100 parameters, as determined by a non-competitive ELISA described herein, and LDL cholesterol levels in a 60 human plasma sample.

Figure 8 is similar to Figure 7 and depicts a significant correlation, r = 0.92, between apo B-100 parameters, determined by the competitive ELISA method of this invention, and LDL cholesterol parameters from the same 60 human samples.

Figure 9 depicts a significant correlation, r = 0.92, as determined by the Spearman Rank Correlation test, of results obtained using a non-competitive ELISA (ordinate) and competitive; Between the results obtained using the ELISA (abscissa) with the same 60 human samples used in Figures 7 and 8.

I. General £ A. Definitions

The term "antibody" refers to a receptor molecule that is a member of a family of glycosylated proteins called immunoglobulins that can specifically associate with an antigen.

An "antibody binding site" is that structural portion of an antibody molecule that encapsulates heavy and light chain variable and hypervariable regions that specifically bind antigen.

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The word "antigen" has been used throughout time to denote the entity that is bound by the antibody and also to denote the entity that induces the production of the antibody. Current practice limits the purpose of an antigen to the entity that is bound by the antibody, while the word "immunogen" is used to refer to the entity that induces the production of the antibody. When the entity used in this context is both immunogenic and antigenic, it is generally referred to as an antigen.

"Antigenic determinant" refers to the actual structural portion of an antigen that immunologically binds to the binding site of an antibody, and is also used interchangeably with the "epitope."

The term "biologically active" refers at least to the ability to specifically bind a ligand or a specifically binding agent, although other general or enhancing properties may also be present.

The word "complex", as used herein, refers to a product formed when a specific binding agent binds a target ligand. Typical complexes include immunoreactants, protein A bound to the antibody, and the like.

"ELISA" refers to an enzyme-linked immunosorbent assay that uses an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify an antigen or antibody in the sample. A description of the ELISA technique can be found in Chapter 22, 4th Edition of Basic and Clinical Immunology, by D.P. Sites et al., Published by Lange Medical Publications of Los Altos, CA in 1982, and U.S. Patent 3,654,090; 3,850,752; and 4,016,043, all of which are incorporated herein by reference.

An "enzyme" refers to a protein that is capable of accelerating or producing, by a catalytic action, some change in a substrate to which it is often specific.

9271 4 18 An "epitope" refers to a portion of a molecule that is specifically recognized by an antibody binding site. It is also referred to as a determinant or antigenic determinant.

"Idiotopes," or "idiotypic determinants," are antigenic determinants located on variable and hypervariable portions of an antibody molecule that can be recognized by the connecting site of other antibodies. Idiotopes are usually divided into two types, those that are binding, site-specific determinants, and those that are non-binding, site-specific. The set of idiotopes on an antibody molecule forms its idiotype. It is assumed that the antibody binding site produced by the hybridoma contains a simple, peculiar set of idiotopes; i. the specific binding site of the antibody to the idiotype.

"Immunoreactant", as used herein, refers to the product of an immunological reaction; i. to the entity formed when the ligand is immunologically bound by the receptor molecule. An immunoreactant is a type of "complex".

The word "separate," as used herein in connection with receptor molecules, means that essentially only one: the species of antibody binding site is lasna.

The terms "labeling agent", "indicating group" or "label" are used interchangeably herein and include simple atoms and molecules involved in the production of either directly or indirectly a detectable signal to indicate the presence of an immunoreactant. Any labeling agent can be attached to or included in the receptor or used separately, and those atoms or molecules can be used alone or in combination with other reagents. Such indicator groups or labels are themselves well known in immunochemistry and form part of the present invention only in pigs when otherwise used with novel receptors, methods and / or systems.

19 92714 "Ligand" refers to a molecule that contains a structural moiety that is bound by a particular receptor, e.g., an antigen that is bound by a receptor.

The term "receptor" is used herein to indicate a biologically active molecule that immunologically binds to (or with) an antigen. Such binding typically occurs at an affinity of about 10 to about 10 ° per liter (M ~), and is the specific interaction of an antigenic epitope with a binding site specific for a receptor antibody.

The receptor molecule of the present invention is any untreated antibody, substantially untreated antibody, or an idiotype-containing polypeptide portion (e.g., a Fab fragment) of an antibody-specific binding site contained in an antibody, such as in a peritoneal fluid or tissue culture supernatant.

The word "receptor" is also used herein to refer to molecules on the cell surface that bind to other molecules. In this context, cell surface receptors are always referred to as the bound entity, preceded by the word "receptor" to avoid ambiguity. A typical cell surface "receptor" is previously; min described LDL receptor.

The biological activity of the receptor molecule becomes apparent in the immunological reaction of the receptor with its antigenic ligand by mixing them together in an aqueous medium to form an immunoreactant, at least at physiological pH values and ionic strengths. The biological activity preferably occurs under biological assay conditions: i.e. under conditions in which the receptor molecules of this invention bind to the antigenic ligand in the pH range of about 5 to about 9, at ionic strengths ranging from the ionic strength of distilled water to about one molar sodium chloride ionic strength, and at temperatures from about 4 ° C to about 4 ° C. 45 ° C. All receptor molecules described herein were biologically active.

The polypeptide portions (antibody binding sites) contained in the idiotype of the antibody binding site of antibodies are those portions of antibody molecules that contain binding site idotopes and bind to a ligand, and include the Fab,

Fab ', F (ab') 2 and F (ab 1) 2 moieties. The Fab and F (ab ') 2 moieties contained in the antibodies are well known in the art and are prepared by the proteolytic reaction of papain and pepsin, in that order, with substantially intact antibodies by methods well known in the art. See, e.g., U.S. Patent 4,342,566 to Theofilopolous and Dixon.

Fab1 antibody moieties are also well known and are produced from F (ab 1) 2 moieties by reducing the disulfide bonds connecting the two heavy chain moieties with e.g. mercaptoethanol and alkylating the resulting protein with a mercaptan reagent such as iodoacetamide. Untreated antibodies are preferred and are used in conjunction with Fab moieties to characterize the monoclonal receptor molecules of this invention.

The words "excrete" and "produce" are often used interchangeably. in the field of cells from which antibody molecules are obtained.

However, cells that produce antibodies may not secrete those molecules into their environment. In this context, hybridoma cells of interest secrete monoclonal antibodies into their environment. Nevertheless, such cells are often referred to herein as "antibody-producing" cells, and their antibodies are said to become "produced", while remaining as used in the art.

The term "specific binding agent", as used herein, refers to a molecular entity capable of selectively binding a ligand. Typical specific binding agents are receptors, comonomer bodies, protein A

and the like.

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The term "substantially pure", as used herein in relation to receptor molecules, means that within the limits of detectability, only one antibody binding site is present as an effective apo B-100 binding agent. Thus, although a substantially pure receptor molecule preparation vox contains more than one species of antibody binding site, such a preparation exhibits a single apo B-100 binding affinity. For example, the tissue culture supernatants produced by the hybridoma of this invention typically contain myeloma proteins as well as the receptors of this invention. A receptor molecule in substantially pure form is typically referred to as a "monoclonal antibody" by those skilled in the art because such compositions are produced using monoclonal hybridoma cultures.

The term "substantially simultaneously" as used herein with respect to mixing 3 or more antigenic and receptor components to form an immunoreaction mixture means that all components are present and mixed together as a single mixture within about 15 minutes, and preferably within about 5 minutes. as a mixture of any 2 components.

B. Hybridomas and monoclonal receptors. The present invention contemplates a hybridoma, labeled HL130C2,3C5, that produces receptor molecules that: (a) immunoreact with a conserved antigenic determinant on the surface of apoprotein B-100; (b) competitively inhibit the binding of apoprotein B-100 to the LDL receptor; and 9 (i) (c) contain an affinity constant for 3.82 x 10 M LDL in a liquid phase competitive equilibrium radioimmunoassay (RIA). The female receptor molecule is commonly referred to as MB47.

Also contemplated in accordance with the present invention is a hybridoma having the laboratory designation V82A6,1G4, which produces receptor molecules that immunoreact with the antigenic determinant of apoprotein B-100 and that contain a 9 -1 af f mutation constant of about 3.0. x 10 M LDL in solid-phase competitive equilibrium RIA. These receptor molecules are commonly referred to as MB24.

Hybridomas HL130C2,3C5 and V82A6,1G4 were deposited with the American Type Culture Collection (ATCC), Rockville, MD on March 6, 1985 under the following ATCC accession numbers:

Hybridoma Receptor ATCC Deposit Number _ Entry___ V82A6,1G4 MB24 HB 8742 HL130C2,3C5 MB47 HB 8746

The above ATCC deposits were made in accordance with the Budapest Treaty (the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure).

The hybridomas of the present invention were formed by fusing an antibody-producing cell and a myeloma cell line. Cells producing such receptors were first described by Kohler and Milstein, Nature, 256, 495 (1975), the disclosure of which is incorporated herein by reference. Receptors are typically obtained from hybridoma cell culture supernatants, preferably monoclonal cell cultures, or alternatively from peritoneal fluid or other body fluid, from non-human, warm-blooded host cells, preferably those that are histologically compatible and immunologically compatible, or immunologically compatible.

In another embodiment of the present invention, there is provided a cell culture of such olives that encapsulates (a) a hybridoma of the present invention; (b) receptor molecules secreted by the hybridoma, which molecules react immunologically

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• «9271 4 23 apoprotein with B-100; and (c) for hybridoma of the culture medium. Media useful for preparing these compositions are both well known in the art and commercially available, and include synthetic media, cultured mice, and the like. A typical synthetic vessel is Dulbecco's minimal vessel / DNEM; Dulbecco et al., Virol. 8, 396 (1959) supplemented with 4.5 g / l glucose, 20 mg glutamine, and 20% fetal calf serum. A typical bred (processed) mouse strain is Balb / c.

In yet another embodiment, the present invention contemplates receptors labeled MB47 and MB24 produced by hybridomas labeled HB 8746 and HB 8742, respectively, that immunoreact with apoprotein B-100. The receptor of the present invention can thus be prepared by culturing a suitable hybridoma of the present invention in a suitable medium, and recovering the receptor from the medium.

Curtiss et al., J. Biol. Chem., 257, 15213 (1982) previously reported the production and characterization of a receptor molecule specific for 11 apo B, including the MB24-labeled resorotor molecule produced by hybridoma HB 8742. Hybridoma HB 8742 was obtained as described in more detail Materials • and Methods by fusing spleen cells from mice immunized with human VLDL.

The IgG fraction of MB24 containing intraperitoneal fluid, formed from intraperitoneal growth of HB 8742, was characterized by isoelectric focusing (IEF). As noted by Curtiss et al., Supra, fusion was performed with P3x63Ag8 myeloma cells secreting IgG1k immunoglobulin. Therefore, in the context of the IEF, a peculiar distribution pattern with multiple protein bands was observed, in which heavy and light chain immunoglobulin molecules P3x63Ag8 myeloma ·· IgG ^ k antibody and MB24 receptor were present when mixed intact.

9271 4 24

Hybridoma HB 8746 produces MB47 receptor molecules and was formed by fusing spleen cells from mice immunized with LDL and P3x63Ag8,653.1 myeloma cells. This variant of the myeloma strain does not secrete myeloma protein.

The intraperitoneal fluid of HB 8746 reveals a special distribution pattern containing protein bands, revealing IgG2a heavy and kappa light chains. Both hybridomas of this invention can thus be characterized in part by the IEF type of the receptor molecules they produce.

Although the V82A6.1G4 hybridoma of this invention produces more than one type of receptor molecule, the receptor molecules of this invention can be readily identified and isolated based on their individual ability to immunoreact with apo B-100 antigenic determinants. The antigenic specificities of MB24 and MB47 were examined by determining their unique ability to immunoreact with apoproteins obtained from chylomicrons, VLDL, LDL, and HDL in a Western blot assay described below.

The results thus obtained showed that MB47 and MB24 immunoreacted with apo B-100 from LDL, VLDL and chylomicrons, but not with apo B-48 from VLDL or chylomicrons. The ability of both MB47 and MB24 to immunoreact individually with apo B-100 from chylomicrons and VLDL is shown in Figure 1.

1. Characterization of the antigenic determinant of apo B-100 immunologically bound by MB47_

Previous studies have shown that apo B-100 is antigenically heterogeneous. This means that some epitopes of apo B-100 are not expressed in all LDL particles.

Mixing with an excess of certain monoclonal antibodies in a liquid phase RIA did not result in immunological binding of all 12 radiolabeled LDL (J-LDL) particles.

• «11 25 9 2 7 1 4

To determine whether the epitope recognized by the MB47 receptor molecules was uniformly expressed by all LDL, the ability of MB47 to immunologically bind to 'J-LDL in a liquid phase RIA was examined. LDL isolated from pooled plasma of 10 normal individuals and plasma from one normal individual was radiolabeled as described below and mixed with biologically active MB47 receptor molecules to form an immunoreaction mixture. The mixture was maintained under biological assay conditions for a predetermined period of time sufficient for MB47 receptor molecules to immunologically bind to apo B in each sample and form an immunoreaction product (immunoreactant).

........ 125

The maximum amount of J-LDL bound by excess MB47 receptor molecules was determined by precipitating all receptor molecules

Using IgSORBr (The Enzyme Co., Boston, MA), and quantifying 125 I-LDL-associated imouls precipitating in a gamma counter.

The results, expressed as trichloroacetic acid (TCA), precipitate 125. .......... .., as a percentage of J-LDL shown in Figures 2, 125, indicate that the antibody bound substantially all of J-LDL; which indicates that all LDL particles express an epitope pin recognized and bound by MB47.

2. Competitive inhibition of Apo B-100 binding to the LDL receptor by MB47

Apoprotein B-100 is the major apoprotein in human and other mammalian LDL and complains of LDL binding to the fibroblast LDL receptor. Previous studies have described the central role of the LDL receptor in mammalian lipoprotein metabolism. The fact that LDL particles isolated from many different animal species bind specifically to the human LDL receptor binding region means that the site of attachment to the apo B-100 molecule must be species-conserved and thus expressed by LDL particles from all species.

• · 26 9271 4

To investigate whether MB47 receptor molecules immunoreact with an antigenic determinant located in the LDL receptor binding region of human aoo B-100, the ability of MB47 to inhibit 125 I-LDL binding to the cellular LDL receptor was studied. This was accomplished by mixing MB47 receptor molecules, in the form of total antibody molecules, with J-LDL to form an immunoreaction mixture. The immunoreaction mixture was then maintained under biological assay conditions for a predetermined period of time sufficient for the MB47 receptor molecules to immunologically bind to plasma J-LDL and form an immunoreactant.

Subsequently, the immunoreaction mixture containing the immunoreactant was applied as a layer on top of human fibroblasts expressing fibroblast LDL receptors. Cultures were maintained for a predetermined period of time sufficient for fibroblast LDL receptors to specifically bind to all LDL receptor 125 binding sites available in the J-LDL-MB47 immunoreaction product. It is believed that such cellular receptor-LDL interaction is inhibited if receptor molecules, as exemplified by MB47, immunoreact with an apo B-100 antigenic determinant, the structure of which is also involved in LDL receptor binding.

The results of this study, shown in Figure 3, indicate that MB47 receptor molecules inhibit human 125 .....

Cell-selective binding, uptake, and degradation of J-LDL by human fibroblasts to a level comparable to that which produces a 200-fold excess of unlabeled LDL.

To investigate the possibility that MB47 receptors bind to an epitope located adjacent to the apo B receptor region and, due to their size, sterically inhibit the binding of apo B to the LDL receptor, the ability of Fab fragments of MB47 antibody molecules to inhibit human LDL the introduction of. and degradation was also examined in the fibroblast assay described above.

π: 27 9271 4

As shown in Figure 4, MB47 Fab fragments significantly inhibited LDL-specific cellular binding and degradation.

Because MB47 Fab fragments are significantly smaller than intact MB47 antibody molecules, it is believed that the epitope recognized by the MB47 antibody attachment site is contained within the LDL receptor binding region on the surface of LDL apo B-100. Antibody MB24 does not have the property of antibody MB47. 125 females and does not inhibit J-LDL binding 3a degradation by cultured fibroblasts.

3. Steric inhibition

For some embodiments of the assay method of this invention, the first and second receptors must bind to different epitopes of the apo B-100 molecule, and their epitopes must be sufficiently distinct that binding of one receptor does not sterically prevent binding of the other receptor. Therefore, the ability of MB47 and MB24 to competitively inhibit each other's immunological binding to the solid phase-fixed apo B-100 reagent was studied.

The results of that study, shown in Figure 5, show that a 70-fold excess of unlabeled MB24 did not significantly inhibit the binding of peroxidase-labeled MB47 to apo B-100 reagent. Similarly, a 70-fold excess of unlabeled MB-47 did not significantly inhibit peroxidase-labeled MB24 from binding to apo B-100 reagent. MB24 and MB47 thus bind to different epitopes on the surface of apo B-100, and those epitopes are sufficiently separated that MB24 and MB47, as intact antibodies, do not inhibit each other's binding to a single apo B-100 molecule.

4. Stoichiometry and affinity of MB47 for binding to apo B-100_

To determine the number of antigenic determinant sites per apo B-100 molecule in LDL identified by MB47 receptor molecules, antibody-labeled RIA was used. In this assay, MB47 receptor molecules were purified from 28,971 4 • 12 5 (isolated) from peritoneal fluid, and radiolabeled (J-MB47) by well-known methods, which are described in more detail in the Materials and Methods section.

........ 125. ..

As shown in Figure 6A, growing Maaria J-MB47 was mixed with solid ground apo B-100 in the form of LDL in separate reaction mixtures. The mixtures were maintained under biological 125 assay conditions for a predetermined period of time sufficient for J-MB47 receptor molecules to immunologically bind to apo B-100 (LDL) and form an immunoreactant. immunoreactivity

the lasna presence of the tant was then determined by precipitating LDL

(bound and free) quantitatively using rabbit antiserum specific for human LDL and expressing immuno- 125 ......

the amount of J-MB47 present as a reactant by gamma counting.

The specific immunological binding of J-MB47 receptor molecules was saturable, as shown in Figure 6A. In addition, the Scatchard curve of the binding results obtained in these studies was linear (Fig. 6B), indicating the homogeneity of the MB47 binding sites on the LDL particles.

In addition, the apparent affinity constant (Ka) of MB47 for human apo B-100 in the form of LDL as determined by antibody-labeled RIA was found to be 3.82x10 M. Scatchard analysis also showed that a maximum of 212 fmoles of J-MB47 antibody bound to 182 fmoles of LDL, indicating that only one MB47 molecule binds to each apo B-100 molecule. This means that MB47 binds to a single, specific antigenic determinant of apo B-100.

The mean affinity constant of MB47 for apo B-100 was also determined in the antigen-labeled RIA described above.

In this competitive equilibrium liquid phase RIA, unlabeled apo B-100 present as LDL caused complete displacement of 125 J-LDL. The calculated affinity constant for MBL of MB47 receptor molecules, which were 9-1 as a whole, intact antibody, was 4x10 M, which

II

9271 4 29 was in good agreement with the Ka determined with the antibody-labeled assay previously described.

C. Methods of determination

The receptor molecules of the present invention are particularly useful for determining the presence and amount of apoprotein B-100 in a body fluid sample such as blood, serum or plasma.

In one embodiment, in accordance with the present invention, there is provided a method of determining the amount of apoprotein B-100 in a body fluid sample, the method comprising the steps of: (a) Producing a sample from the body for determination. Such a sample is usually taken as a measured amount or a known amount of blood, and more preferably as plasma or serum. Methods for taking blood, plasma and serum samples are well known in the art and will not be further processed in this context.

(b) Producing receptor molecules in a biologically active form that (i) immunoreact with apoprotein B-100 and (ii) are secreted by either a hybridoma having an ATCC accession number of HB8746 or a hybridoma having an ATCC accession number of HB 8742, and which is lasna as an effective means of performing the determination.

In preferred embodiments, the receptor is an intact antibody or Fab fragment.

The effective number of receptor molecules may vary, depending, inter alia, on the particular assay method used, as is well known. Well known is also the ease with which an effective amount can be determined using standard laboratory technology by one skilled in the art.

(c) Mixing the body fluid sample with the receptor molecules of step (b) to form an immunoreaction mixture.

30 92714 (d) The mixture is maintained under biological assay conditions for a predetermined period of time from minutes to hours, such as from about 10 minutes under biological assay conditions to a predetermined time of about 16-20 hours sufficient to immunologically bind apoprotein B to the antibody binding sites of the receptor molecules: to form an immuno-reactant (first complex). Biological assay conditions are those that maintain the biological activity of the receptor molecules of this invention and are characterized by a temperature range of about 4 ° C to about 45 ° C, a pH range of about 5 to about 9, and a variation in ionic strength from distilled water to about one molar. sodium chloride counterpart. Methods for optimizing such conditions are well known in the art.

(e) Determine the amount of total immunoreactant formed and thus the amount of apo B-100 present in said sample.

In preferred embodiments, the body fluid sample of step (a) is further prepared from the assay according to step (e) via the following steps: (f) Producing biologically active second receptor molecules secreted by portions of each hybridoma of step (b); (g) Mixing a predetermined amount of other receptor molecules with the body fluid sample to form an immunoreaction mixture.

(h) Maintaining the second receptor / body fluid sample thus formed in a manner similar to that described in step (d) to form a layered immunoreactant (second complex) comprising one MB47 molecule and one MB24 molecule immunologically linked to apo B-100. molecule.

The general configuration methods described above can be performed, as is well known in the art, using many different formats.

II

31 9271 4 Thus, in contrast to the use of solid phase formats in the more specific assay methods described below, the invention is not so limited.

Solid phase assay formats can be implemented using either receptor molecules or antigen attached to a solid matrix to form a solid support. In those embodiments where the solid support comprises the receptor from step (b), the mixture from step (c) is a solid / liquid phase mixture, and the immunoreactant from step (d) is a solid phase immunoreactant containing the body sample apo B-100.

In those embodiments where the solid support comprises the apo B-100 reagent, the mixture from step (c) is also a solid / liquid mixture, but the immunoreactant from step (d) containing the body sample apo B-100 is a liquid phase immunoreactant. Reagent apo B-100 is biologically active; i. antigen, apo B-100, supplied from a source other than the one under study, typically in the form of isolated LDL.

Determination of the amount of Apo B-100 bound as an immunoreactant in step (d) can be accomplished, directly or indirectly, by assay methods well known in the art. For example, homogeneous determination systems, such as those described in U.S. Patent Nos. 4,536,479, 4,233,401, 4,233,402, and 3,996,345, all of which are incorporated herein by reference, may be used.

In preferred solid phase embodiments, the body fluid is further prepared from the assay using a labeled specific binding agent. The type and specificity of the labeled specific binding agent will depend, as is well known in the art, on the method and format used.

In preferred solid phase embodiments, wherein the solid support comprises a receptor from step (b); that is, the receptor of this invention, the amount of apo B-100 bound to the solid phase is prepared from the assay by the following steps: 9271 4 32 (i) Producing biologically active labeled second receptor molecules that bind to the apoprotein B-100 in the body sample. to form an immunoreactant. The label of the labeled second receptor is capable of detecting the presence of a labeled second receptor in the immunoreactant.

In particularly preferred embodiments, the labeled second receptor molecules react immunologically with another apo B-100 epitope that is different from the epitope with which the solid-phase receptor molecules react or does not significantly inhibit the solid-phase receptor molecules from reacting with apo B. The labeled second receptor molecules are those secreted by the fractions from each of the hybridoma steps (b); i. listed receptor molecules that were not selected as the first receptor molecules.

Methods for determining whether a receptor molecule inhibits (interferes with) the binding of another receptor molecule to the same antigen are well known in the art and are described in more detail below.

(j) Mixing a predetermined amount of labeled second receptor molecules with the body fluid sample to form an immunoreaction mixture.

The mixture thus formed may be a liquid mixture, such as when step (i) is carried out before step (b) above, or it may be a solid / liquid mixture when the labeled second receptor is mixed substantially simultaneously with or after step (b). When step (i) is performed before or substantially concurrently with step (b), the labeled second receptor molecules immunoreact with another apo B-100 epitope that is different from the epitope to which the solid-phase receptor molecules react or substantially inhibit the solid phase. bound receptor molecules from immunoreacting with apo B-100.

li 33 92714

In preferred embodiments, step (i) is performed substantially simultaneously with or after step (b).

(k) The labeled second receptor / body fluid sample mixture thus formed is incubated in the same manner as described in step (d) to form an immunoreactant.

The solid phase-bound receptor molecules and other receptor molecules thus immunologically bind to apo B-100 in the body fluid sample, thus forming a solid-phase-bound layered immunoreactant containing a label bound to it. That is, a solid-phase layered immunoreactant containing a label is formed when one molecule of apo B-100 immunoreacts with both a solid-phase-bound receptor molecule and a labeled other receptor molecule. In preferred embodiments, all labeled second receptor molecules that do not form part of a solid phase bound immunoreactant (i.e., those that are not immunologically bound to apo B-100, which itself binds to solid phase receptor molecules) are separated from the immunoreactant by immunoreactant, preferably by washing. determination of the amount of labeled second receptor.

The determination of the amount of immunoreactant formed according to step (e) is performed as part of the determination of the amount of labeled second receptor bound to the immunoreactant, which immunoreactant contains apo B-100. This provides a direct determination of the amount of apo B-100 contained in the sample.

That number may be zero, thus indicating that there is no apo B in the sample, within the limits that can be expressed. Methods for determining the amount of labeled second receptor will depend on the label used, such labels and methods of determination being well known in the art.

In preferred solid phase embodiments where the solid support comprises apo B-100 reagent, the amount of immunoreactant formed in step (d) is prepared from the assay by the following steps: (1) Mixing a biologically active labeled specific binding agent, preferably a receptor, -t \ m to any of the receptor molecules used in step (b) which are lasna as a solid phase immunoreactant to form a complex, preferably another immunoreactant. The label of the labeled specific binding agent begins to indicate the presence of the labeled specific binding agent in the complex. In preferred embodiments of these types of assays, steps (i) to (k) are performed after step (d).

(m) Mixing a predetermined amount of labeled specific binding agent with a body fluid sample to form a reaction mixture, preferably an immunoreaction mixture.

(n) The labeled specific binding agent / first immunoreactant mixture thus formed is incubated in the same manner as described in step (d) to form a complex.

The solid phase complex thus formed contains a label bound into it. In preferred embodiments, any labeled specific binding agent that is not partially bound to the solid phase complex is separated from the complex, preferably by washing, prior to determining the presence of the label bound to the solid phase.

The determination of the amount of immunoreactant formed according to step (e) is carried out by determining the amount of label present as part of the complex. This provides an indirect determination of the amount of apo B-100 in the sample in the sample.

Labeling of protein antigens and specific binding agents is well known in the art. For example, receptors produced by hybridomas can be labeled by metabolic linking of radioisotope-containing amino acids

II

9271 4 35 as a component in a tissue culture medium. See, e.g., Galfré et al., Meth. Enzymol. 73, 3-46 (1981).

Protein conjugation or coupling methods via active functional groups are particularly useful, and result in the label being covalently linked to the antigen or specific binding agent. See, e.g., Aurameas, et al., Scand. J. Immunol.

Vol. 8, Suppl. 7, 7-23 (1978) and U.S. Patent 4,493,795, which is incorporated herein by reference. In addition, a site-directed coupling reaction can be performed such that the label does not substantially interfere with the biological activity of the antigen or receptor, e.g., Rodwell et al., Biotech. 3, 889-894 (1985).

The labeling agent can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form fluorochrome (copper), a useful immunofluorescent marker. Suitable fluorescent labels include fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), 5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), rhodamine (8), lysamine and the like. A description of immunofluorescence analysis methods can be found in DeLuca, "Immunofluorescence Analysis", Antibody As A Tool, Marchalonis et al., Edit. J. Wiley & Sons, Ltd., pp. 189-231, 1982, which is incorporated herein by reference.

In preferred embodiments, the indicating group is an enzyme such as horseradish peroxidase (HRPO), glucose oxidase, or the like. When the main detecting moiety is an enzyme such as HRPO or glucose oxidase, additional reagents are needed to visualize the fact that a receptor-ligand complex (immunoreactant) has formed. Such additional reagents for HRP0 include hydrogen peroxide and, s 9271 4 ό Ο an oxidizing copper precursor such as diaminobenzidine. An additional reagent useful with glucose oxidase is 2,2'-azino-di- (3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS).

Radioactive elements are also useful labeling agents and are illuminating in this context.

A typical radiolabel is a radioactive element that produces gamma-rain emissions. Elements that themselves emit

,. ·, ... 124 T 12 5 128 131 T 132T

gamma rains such as J, J, j, J, J

and ^ Cr, represent one class of radioactive element-containing detecting groups producing gamma-rain emission.

125

Particularly preferred is J. another group of useful indicating groups contains those elements, such as ^ C, ^ F, ^ O and N, which themselves emit positrons. The positrons thus emitted produce gamma rains when they encounter electrons in the living body. Also useful is a beta emitter like 111.

indium.

Thus, the assay methods and systems of the present invention may utilize or encapsulate the receptor of the present invention attached to a solid matrix to form a solid support.

The antigen or receptor is typically attached to a solid matrix by adsorption from an aqueous medium, although several methods of adsorption, as well as other methods of attachment well known to those skilled in the art, may be used. Typical such methods are the reaction of a receptor or antigen with a reactive carboxylic functionality formed by the reaction of cyanogen bromide with glucose-containing matrices such as, e.g., cross-linked dextrose or cellulose, as a glutaraldehyde linker, as discussed below in the case of a co-latex.

Useful solid matrices are well known in the art. Such materials include cross-linked dextran, which is

II

37,92714 available under the tradename Sephadex Pharmacia Fine Chemicals (Piscataway, NJ); agarose; polystyrene spheres, about 1 to about 5 mm in diameter, available from Abbott Laboratories of North

From Chicago, IL; polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose or nylon-based fabrics such as sheets, strips or rods; or tubes, plates, or wells of a microtiter plate, such as those made of polystyrene or polyvinyl chloride.

Latex particles, which are useful in agglutination-type assays, are also useful solid matrices. Such materials are supplied by the Japan Synthetic Rubber Company of Tokyo, Japan, and are described as carboxy-functional particles dispersed in an anionic soap. Typical batches of such particles have an average diameter of 0.308 microns (μ) and contain an average carboxy / 2 functional group distribution of about 15 to about 30 Angstroms per carboxy group.

Prior to use, the particles are reacted with a diamine such as 1,3-diamino-2-propanol to form a large number of amide bonds with the carboxy groups of the particle while leaving the amine groups free. The free amines are then reacted with a dialdehyde such as glutaraldehyde and a receptor or antigen to form Schiff's base reaction products. The reaction products of Schiff's base are then reduced with a water-soluble reducing agent such as sodium borohydride to provide a useful solid support.

Those skilled in the art will appreciate that there are numerous methods for solid phase immunoassays that can be used in this context. Typical, useful solid-phase assays include multiple enzyme immunoassays (EMIT) and fluorescence immunoassays (FIA), especially in addition to treated RIA and ELISA assays. However, any method that results in the detectable reaction of apo-protein B-100 with the 9271,438 receptor molecules of this invention is considered part of this invention. In each of these assays, single or double antibody techniques can be used that use an indicative agent to detect an immunoreaction, and thus to detect binding of all apo protein B-100 to be assayed with the receptor of this invention. Typical methods can be found as described in Maggio, Enzyme Immunoassay, CRC Press, Cleveland, OH (1981); and Goldman, Fluorescent Antibody Methods, Academic Press, New York, NY (1980).

One embodiment of a specific method for determining apo B-100 in a body fluid sample using the solid phase attached receptor of this invention is a non-competitive ELISA characterized in that immunoreactions are performed sequentially. In such an assay, an even amount of MB47 receptors, e.g., generally about 1 to about 500 ug, is attached to the inner walls of the microtiter well to form a solid support.

Any apo B-100 in the plasma taken from the body sample is then immunoreacted with receptors attached to the solid phase. This is accomplished by mixing a known amount, e.g., about 10 to about 200 microliters (yUl) of sample (which can be prediluted), such as serum or plasma, with receptors attached to the solid phase in a microtiter well to form a solid / liquid phase mixture. The mixture is maintained under biological assay conditions for a predetermined period of time sufficient for all apo B-100 in the sample to immunologically bind to the receptor molecules and form a solid phase immunoreactant. The solid and liquid phases are then separated, and the solid phase is typically rinsed to facilitate ensuring the removal of non-specifically bound materials.

Apo B-100, which is lasas as a solid-phase immunoreactant ’(i.e., apo B-100 bound to solid-phase

II

39 9 2 7 1 4 MB47 receptors) are then immunoreacted with other enzyme-labeled receptor molecules. This is accomplished by mixing a predetermined amount, e.g., about 0.1 to about 10 ug of enzyme-labeled second receptor molecules in an aqueous buffer solution, preferably horseradish peroxidase (HRPO) -labeled MB24 receptors, to form a second solid / liquid mixture. The second mixture is incubated as described above to form a solid phase immunoreactant "sandwich" containing the MB47 receptor, apo B-100 and enzyme-labeled MB24 receptors.

After separating the liquid phase from the solid phase as described above, an equal chromogenic substrate such as o-phenylenediamine (OPD) for HRPO is mixed into a microtiter well containing a solid phase immunoreactant to form a third solid / liquid mixture. This mixture is maintained under the conditions of the biological assay for a predetermined period of time sufficient for all the enzyme in the immunoreactant to convert the substrate of the corresponding earth into a colored product. The optical density of the resulting colored solution is then measured and compared with the results obtained using solutions containing the known Maaria apo B-100 reagent.

The ability of the non-competitive, periodic ELISA, described and described in more detail above in the Materials and Methods section, to detect apo B-100 contained in a body fluid sample, was examined. In those studies, equal amounts of human lipo-protein-free plasma (LPD) mixed with known Maaria apo B-100 reagent served as a body fluid control.

The results of that study, shown in Table 1 below, show that the method described above can accurately determine the clinically relevant Maari of apo B-100 in the body fluid sample.

• 9271 4 40

table 1

Non-competitive, periodic ELISA studies of apo B-100 in human plasma ___

Example1 True Ref.1 MB47 / MB242 MB24 / B183 nen2 _ _ B low 40 32 + 44.3 42.6 + 5.0 31.7 B normal 80 71 + 103 82.3 + 11.8 54.7 B high 150 126 + 180 149 + 24.7 119 T low 22.3 12.6 + 17 16.2 + 0.62 15.4, 9.0 T normal 44.8 25.4 + 49.2 36.1 +9.4 22.9, 19.7 T high 89.5 64.0 + 101 77.4_ + 7.4 34.9: 36.3 A 73 82.9 + 11.7 66.4 AM 76 67 , 1 + 8.1 46.8, 61.0 B 86.5 69.8 + 4.6 68.5, 90.5 C 145 129 + 3.8 147.173 D 109 81.0 + 7.4 76, 3: 98.2 K 95 98.4 + 16.5 77.5, 88.9 r 60 64.2 + 7.5 51.3, 52.9

Collected 81.3 78.5 + 6.8 54.7, 65.9

Cl 40.1 47.9 + 6.7 47.0, 68.7 C2 74.8 88.6 + 13.2 81.3, 98.2 C3 70.4 64.5 + 4.8 64.5 ; 82.9 C4 39.9 56.7 + 6.6 48.5; 58.0 ^ Control solutions containing clinically low, normal, and high apoprotein B-100 were obtained from Johnson & Johnson Biotechnology Center Inc., La Jolla. , CA (B) and Tago Inc., Burlingame, CA (T). Plasma samples were obtained from clinically normal monopolies (one or two letter markings and C1-C4), and the pool represents a mixture of 10 such samples.

2 ...

Actual apoprotein B-100 concentration as determined by total protein added to the sample. All concentrations shown in the table are in mg / dl.

li Λ ...

2

The concentration of apoprotein B-100 as determined by 3 is used from the reference assay (ref) subsequently depicted. Values are given in the range of three standard deviations.

4 9271 4 41

Apoprotein B-100 concentrations (mean + 3 standard deviations) obtained using solid phase-attached MB47 receptors and HRPO-labeled MB24 receptors were not in a competitive periodic ELISA as described previously.

Apoprotein B-100 concentrations (reproducibility values shown when made) obtained using solid phase-attached MB24 receptors and HRPO-labeled B18 receptors and / Curtiss et al. , J. Biol. Chem., 257: 15213-15221 (1982J7 in a non-competitive ELISA in which immunoreactions were performed extensively after the solid phase immunoreactant was first formed.

Another embodiment of a specific method for determining apo B-100 in a body fluid sample is a non-competitive ELISA, characterized in that the immunoreactions are performed substantially simultaneously. The microtiter well described above, which contains MB47 receptors attached to the solid phase, is mixed with a substantially co-taken body sample and enzyme-labeled other receptors that immunoreact with another apo B-100 epitope and do not substantially inhibit MB47 receptor binding. Loo of. Such enzyme-labeled second receptors are preferably MB24 receptors.

The solid / liquid mixture formed is then incubated, the phases of the mixture are separated, and the amount of enzyme present in the solid phase bound immunoreactant "sandwich" is determined as previously described.

The non-competitive ELISA described above was used to examine plasma apo B-100 levels in 20 patients with coronary artery disease (CAD), 20 patients with normal familial hypercholesterolemia, and 20 normal individuals. As shown in Table 2 below, the mean plasma apo B-100 level in normal individuals was defined as 85 milligrams / deciliter (mg / dl) 2 in the standard deviation range of 9271 4 42 + 21 mg / dl. This value is in good agreement with the normal range of apo B-100 values reported by Curry et al., Clin. Chem. 24, 280-286 (1987), and Rosseneu et al., Clin. Chem. 28, 427-433 (1983), each of which used different immunoassays.

Further, plasma apo B-100 levels in patients with CAD and hypercholesterolemia were higher than the upper limit of the 2 standard deviation ranges normally found. Similar results, using other methods in patients with CAD and hypercholesterolemia, have been reported by Sniderman et al., Proc. Natl. Acad. Sci.

USA 77, and Lipid Research Council, JAMA 251, 351-364 (1984).

Table 2

Simultaneous ELISA tests of apo B-100 in human plasma 1 .2 ... 3

Single-state Lipoprotectant levels_ Apoprotectant B-levels___

Size 3 HDL4 LDL1 2 Kompet® Ei- η RID8 RID9 col. kol. kol. ELISA Competent # 1 # 2 _____ __ ___ _____ ELISA _____ ____

Normal 186 60 111 85 82 102 69 + SD 37 16 30 21 22 20 19 CAD 205 37 133 109 104 138 84 + SD 35 7 37 24 23 20 24 FH 331 37 270 196 204 211 136 + SD 90 11 96 40 49 64 44

Monocles include 20 normal lipid blood, healthy controls (normal); 20 individuals with coronary artery disease as defined by cardiac catheterization (CAD); and 20 individuals with normal hypercholesterolemia (FH).

2. . .

The values given are means +2 standard deviations from the mean; (+ SD) in mg / dl.

3

Total cholesterol.

4. ....

Cholesterol, which is lasna as HDL.

I! 2

Cholesterol, which is lasna as LDL.

„92714 4 3 ^ Apo B-100 level as determined by the competitive ELISA (compet.) Method described in the Materials and Methods section.

7

Apo B-100 level, determined by non-competitive (Non-competitive) ELISA, using solid phase MB47 reseoters and HRPO-labeled MB24 receptors, in which the sample and receptors were mixed substantially simultaneously.

8

Apo B level, determined using the radial immunodiffusion putty model Diffogen RID, available from Tago Inc., Burlingame, CA.

9 .........

Apo B level, determined using the M-Partigen RIA rainfall immunodiffusion kit model, available from Calbiochem-Behring,

La Jolla, CA.

The effect of diluting the body fluid sample, before determining apo B-100 from it by a non-competitive simultaneous ELISA method, was also investigated. Those results, shown in Table 3 below, show that there was no significant difference in apo B-100 levels as determined in the 5-fold range of plasma dilutions, i. 1: 1000-1: 5000.

Table 3

Simultaneous ELISA studies of Apo B levels determined from different plasma dilutions_

Plasma dilution Plasma 1 ^ Plasma 2 ^ Plasma 3 ^ 1: 1000 54.4 136.6 199.0 1: 2500 57.5 142.5 194.2 1: 5000 53.0 126.0 178.0

Mean2 55.0 135.0 190.0 S.D.3 1.88 6.83 8.98% C.V.4 3.4 5.0 4.7 3 Plasma apo B-100 concentration in mg / dl.

2

Mean plasma apo B-100 concentration for all 3 dilutions.

44 9 2 7 1 4 3

One standard deviation.

4 Coefficient of variation percentage between results obtained for different dilutions.

Figure 7 shows the correlation between LDL cholesterol and plasma apo B-100 levels as determined by non-competitive ELISA for normal and patient samples as described above. The correlation coefficient of 0.89 is similar to that reported by Albers et al., Metabolism 24, 1339-1351 (1975) and Slater et al., Clin. Chem. 31, 841-845 (1985).

Embodiments of the assay methods of this invention using a solid support comprising apo B-100 reagent attached to a solid support are performed using the following steps: (a) A body fluid sample containing apo B-100 is taken as described above.

(b) Stirring substantially simultaneously (1) the liquid sample; (2) a predetermined number of either MB24 or MB47 receptor molecules; and (3) a predetermined amount of apo B-100 reagent attached to the solid phase to form a liquid / solid phase immunoreaction mixture.

The mixture is maintained under biological assay conditions for a period of time sufficient for the antibody binding sites of the resorotor molecules to immunoreact (immunologically bind) to either the apo B-100 reagent or all of the apo B-100 present in the body fluid sample. Receptor molecules that immunologically bind apo B-100, which is present in the body fluid assay, form a liquid phase immunoreactant, and those that bind apo B-100 reagent attached to a solid phase form a solid phase immunoreactant.

II :.

* „92714 4 5 (c) The presence of the immunoreactant attached to the solid phase is then determined, and the amount of apo B-100 in the sample is thus determined by comparing the amount of binding that occurs when a known amount of MB47 or MB24 is mixed with a similar solid. phase-attached apo B-100, as subsequently treated. The immunoreactant assay is preferably performed after the liquid phase and solid phase immunoreactants have been separated from step (b), e.g. by washing. The amount of apo B-100 contained in the sample bound as a liquid phase immunoreactant can be determined as using labeled antibodies that immunoreact with the receptor molecules of this invention, such as peroxidase-linked goat anti-mouse Ig, or as otherwise described. .

In a particularly preferred embodiment of the competitive assay, about 1 ug to about 10 ug of apo B-100 reagent is attached to a solid matrix, preferably the walls of a microtiter well, to form a solid support. All non-specific binding sites on the solid support are typically blocked by a protein such as BSA or the like.

The receptor molecules of the invention of the predetermined amount, e.g., generally from about 0.1 to about 10, are subjected to substantially simultaneous immunoreaction with apo B-100 reagent attached to the solid phase and any apo B-100 in the body fluid sample. (which sample can be diluted), such as serum or plasma. This is accomplished by mixing a substantially equal amount of sample and receptor molecules simultaneously in a microtiter well containing apo B-100 reagent attached to the solid phase.

The solid / liquid mixture thus formed is maintained under biological assay conditions for a period of time sufficient for the receptor molecules in the plasma to immunologically bind the apo B-100 in the glass. Solid and liquid phase; it is then preferably separated from one another, e.g. by washing, 46 9 2 7 1 4 τ— and the solid phase is typically rinsed to facilitate the removal of non-specifically bound materials.

The presence of immunoreactants attached to the solid phase is then typically determined using labeled antibodies that immunologically bind to the receptor molecules of this invention, such as peroxidase-labeled goat anti-mouse IgG.

In a co-competitive ELISA, apo B-100 in a patient sample competes with a constant known amount of apo B-100 reagent for a constant known amount of antibody attachment sites for the receptor molecule in the immunoreaction mixture. The competition that apo B-100 in the sample enters upon entry results in a decrease in the detectable solid phase bound immunoreactant; the greater the decrease, the greater the amount of apo B-100 in the body sample in the study.

In order to obtain the relative levels of apo B-100 in plasma plasma, the results obtained using competing standards containing the known Maaria apo B-100 reagent were compared. A standard curve was prepared using LDL concentrations ranging from 32 mg / ml to 0.25 mg / ml (320 mg / dl to 2.5 mg / dl).

The competitive E LIS A described above was used to study the same normal and patient samples that were judged by a non-competitive ELISA. Those results, which are also shown in Table 2 before this, are in good agreement with the results obtained in the non-competitive determination.

The effect of diluting the body fluid sample before determining apo B was examined in the competitive ELISA described above. Those results, shown in Table 4 below, indicate that there were no significant differences in apo B-47 9271 4 levels as determined by the 4-fold plasma dilution range; i. 1: 100-1: 400.

Table 4

Apo B-level studies as determined by competitive ELISA for different plasma dilutions_

Plasma dilution Plasma 1 ^ Plasma 2 Plasma 3 1: 100 32.8 63.3 175.8 1: 200 30.0 58.5 183.2 1: 300 31.8 64.8 190.9 1: 400 33.8 62.6 190.0

Mean2 32.1 62.3 185.0 S.D.3 1.6 2.7 7.0% C.V.4 5.0 4.3 3.8 Plasma apo B-100 concentration in mg / dl.

2

Mean plasma apo B-100 concentration for all 3 dilutions.

3

One standard deviation.

4

The percentage variance between the results obtained for the different dilutions.

The correlation between LDL cholesterol and plasma apo B levels as determined by competitive ELISA with the normal and patient samples described above is shown in Figure 8. The correlation coefficient of 0.92 is similar to that of Albers et al. reported, Metabolism 2j4, 1339-1351 (1975), and Slater et al. reported by Clin. Chem., 31, 841-845 (1985), showing that a competitive ELISA accurately predicts circulating LDL cholesterol levels.

The correlation of Apo B-100 levels, obtained using competitive and non-competitive ELISA, was examined. As shown in Figure 9, there was a high correlation (r = 0.92) between the results 9271,448 obtained in each assay.

The diagnostic system, preferably in the form of a kit useful in performing the above assays, comprises, in separate packages, (a) a first specific binding agent, wherein said agent is (i) a receptor that immunoreacts with apo B-100, and is selected from receptors secreted by hybridoma HB 8746 (MB47) or hybridoma HB 8742 secreted receptors (MB24) and (b) a second specific binding agent labeled to express the immunoreaction of said first binding agent with apo B-100. The labeled specific binding agent is preferably a receptor coupled to the enzyme. More preferably, the labeled agent is a fraction of each of the receptors in (a) above, coupled to an enzyme.

In further preferred embodiments, the system further comprises a second reagent apo B-100 silo for use as a control and / or target antigen. Also preferred are embodiments characterized in that the system comprises a solid matrix to which the first specific binding agent or apo B-100 reagent is attached to form a solid support. Useful solid matrices have already been described. Preferably, however, the solid matrix is a well of a microtiter plate.

Known Maaria specific binding agents are supplied.

Those parameters are sufficient to perform at least one determination. The specific binding agents to be used are typically supplied in a form and amount designed to be diluted to a specified volume of water, saline or a buffer, such as phosphate buffered saline at pH 7.3-7.5.

Other packages may also be included in the system. Such packages may contain (i) buffer salts in dry or liquid form, (ii) enzyme substrates such as o-phenylenediamine, and the like.

li „92714

Typical packages include glass and plastic bottles or containers such as polyethylene and polypropylene; plastic, plastic-metal laminates, plastic-metal laminate-paper shells and the like. The specific binding agents may be packaged in an aqueous liquid form such as peritoneal fluid or buffer, but are preferably delivered in a dried form such as that provided lyophilized.

D. affinity sorbant

The present invention also contemplates an affinity sorbent, preferably sterile, comprising a biologically active receptor of the invention attached to a solid matrix to form a solid support. The solid matrix is preferably in particulate form. Such affinity sorbents are useful for the specific removal of lipoprotein (VLDL and LDL) containing apoprotein B-100 by immunoadsorption from the plasma of patients suffering from hypercholesterolemia.

The solid support can consist of a wide variety of materials, such as cross-linked dextran, e.g., Sephadex G-25, -50, -100, -200 and the like available from Pharmacia Fine Chemicals, Piscataway, NJ, agarose and cross-linked. , e.g. Sepharose 6B, CL6B, 4B, CL4B and the like, also available from Pharmacia Fine Chemicals, or Bio-Gel A-0.5M, A-1.5M, A-50M and the like, available from Bio-Rad Labora -tories from Richmon, CA, or polyacrylamide spheres, e.g. Bio-Gel P-2, P-30, P-100, P-300 and the like, also available from Bio-Rad Laboratories. Agarose and crosslinked agarose materials are preferred in this context due to their high porosity and low non-specific binding properties, and will be used as an illuminating solid matrix.

Sterilization of receptors and solid matrix is usually performed before coupling. To preserve the biological activity, i.e. 92714, the receptors are usually sterilized by filtration, for example by passing through a 0.22 micron nitrocellulose filter. The sterilization of the matrix depends, as is well known, on the type of matrix. For example, Sepharose cannot be sterilized by autoclaving, but can be chemically sterilized, for example by treatment with diethyl pyrocarbonate. On the other hand, cross-linked Sepharose can be sterilized by autoclaving at pH 7, 120 ° C for 20 minutes.

Sephadex or Sepharose is typically activated for coupling using cyanogen bromide by methods well known in the art. The activated matrix is then washed and ligated to receptors that immunoreact with apo B-100 and secreted by hybridoma HB 8746 or hybridoma HB 8742. The receptor attached to the matrix is then washed and ready for use. The unreacted reactive groups on the support can be reacted with an amine such as ethanolamine or Tris, if desired.

The affinity sorbent can be used in a loose state, but is preferably limited to a column. The patient's plasma containing apo B-100 is then mixed with an affinity sorbent to form an immunoreaction mixture. The mixture is maintained under biological assay conditions for a period of time sufficient for the receptors attached to the solid matrix to immunologically bind plasma apo B-100 in plasma and form a solid immunoreactant attached to the matrix. Plasma is then separated from the immunoreactant attached to the solid matrix. The Apo B-100 (LDL and VLDL) -free plasma thus born can then be transferred to the patient from whom it was originally obtained.

The preparation of an affinity sorbent and its use for the removal of lipoproteins containing apolipo protein B from plasma is described by Stoffel et al., Proc. Natl. Acad. Sci. USA 78, 611-615 (1981), which is incorporated herein by reference. An affinity -1 adsorbent column prepared by attaching substantially pure MB47 receptors to CNB4-activated Sepharose 4B was found to immunoadsorb (bind) 3 mg of LDL per ml of solid support.

II. Materials and Methods A. Preparation of human lipoproteins

Human lipoprotein fractions were isolated by centrifugation at the following densities: VLDL, density (d) less than 1.006 g / ml; LDL, d equal to 1.025-1.050 g / ml; HDL, d equal to 1.070-1.21 g / ml, and LDS, d equal to 1.21 g / ml. In some cases, human LDL was also isolated with a d of 1.019-1.063 g / ml or 1.045-1.065 g / ml. The protein content of the plasma and each fraction was determined by the method of Lowry / Lowry et al., J. Biol. Chem.

19 3, 265-275, (1951J_7 as modified by Markwell et al., Anal. Biochem. 8-7, 206-210 (1978), using the BSA standard.

For each LDL and VLDL fraction, the apo B concentration was also estimated after apo B was precipitated with tetramethylurea (TMU) using the method of Kane et al. technology, J. Clin. Invest., 5-6, 1622-1634, (1975) or with isopropyl alcohol

Equsan et al. according to the teachings, J. Lipid Res., 2-4, 1261-1267 (1983).

Fresh, fasted human plasma was obtained from normal healthy donors by plasmaphoresis and obtained at 0.1% EDTA (s / v). An aggregate sample from three or more donors was used unless otherwise noted. Lipoproteins were isolated by intermittent plasma ultracentrifugation using solid KBr to obtain density (d). Lipoprotein fractions included VLDL, d less than 1.006 g / ml; IDL, d = 1.006-1.019 g / ml; LDL, d = 1.019-1.063; and HDL, d = 1.063-1.25 g / ml.

The bottom fraction containing lipoprotein-free serum (d greater than 1.25 g / ml) was also collected.

Fractions were thoroughly dialyzed against lipoprotein buffer containing 0.15 M NaCl, 0.3 mM EDTA, 0.0005 52 9 2 7 1 4% alpha-tocopherol at pH 7.4.

The chylomicron fraction was separated from the VLDL fraction from non-fasted pooled plasma by counting the chylomicrons in lipoprotein buffer by ultracentrifugation at 120,000 x g for 40 minutes at 4 ° C.

All lipoproteins were filter sterilized and stored at 4 ° for up to 20 days. The protein content of lipoproteins was analyzed according to a modification of Lowry's method, J. Biol. Chem. 193, 265-275 (1951), using the BSA standard. All lipo-protein concentrations are expressed based on protein.

The apoprotein composition of each lipoprotein class was determined by SDS-polyacrylamide gel electrophoresis. Chylomicrons contained detectable Marie apoproteins B, E, and C, while VLDL contained apoproteins B, E, C, and small amounts of apoprotein A-I.

IDL contained apoproteins B, E, and C, whereas LDL contained only apoprotein B. HDL contained apoproteins A1, All, and C.

During these studies, lipoprotein preparations appeared to have a uniform apoprotein composition. To control possible proteolysis of apoproteins, selected plasma pooled samples were isolated and stored with 1 mg / ml gentamicin sulfate, 0.2% sodium azide, and 1 mM benzamidine, 10 mM diisopropyl fluorophosphate, 10 μg / ml soybean trypsin inhibitory.

Comparison of the SDS-PAGE apoprotein staining models of these lipoproteins, as well as those isolated without antibiotics and protein inhibitors after ultracentrifugation and storage for 3 weeks, showed no signs of proteolysis. All preparations used were sterile.

• · ·

II

92714 53 B. Hybridoma Production and Culture Untreated native lipoproteins isolated from pooled plasma were used for immunization. Four to five week old Balb / c mice were immunized intraperitoneally with 50 micrograms of lipoprotein in complete Freund's vehicle. Secondary intravenous injections containing 50 ug of lipoprotein in lipoprotein buffer were given between days 28 and 33. Spleens were removed from immunized mice 72 hours after the last injection, and single-cell suspensions were prepared in HT medium. Blood was also collected and serum was used as a positive control for each immunoassay.

Mouse myeloma cell lines were maintained in log phase growth in stationary cultures in complete HT medium / Kennet et al., Curr. Top. Microbiol. Immunol. 81, 77-91 (1978), which is incorporated herein by reference, which contained 0.1 mM azaguanine. Fusions were performed with 30% (v / v) polyethylene glycol 10000 (Sigma, St. Louis, MO) relative to immune spleen cells - P3x63Ag8 10: 1. Three days after fusion, cells were seeded in 96-well tissue culture at a concentration of 1x10 viable cells / well in HT medium containing 0.1 mM aminopterin.

• Cells were given 7 days post-fusion HT medium, and approximately 4-5 days later thereafter as needed.

Growth was monitored microscopically, and culture supernatants were collected at day 14 from the determination of antigen-specific antibody production by solid phase RIA. Specific antibody-producing hybridomas were cloned 19-47 days after fusion by limiting dilution with Balb / c spleen mast cells, and hybridomas in wells containing simple colonies were screened for antibody production by solid phase RIAr. . The cloned hybridomas were cultured in medium containing 10% calf serum and stored frozen in liquid nitrogen.

54 9271 4 C. Inhibition of LDL binding, transport and degradation 125

The ability of anti-apo B-100 receptors to bind, transport and degrade mhiboxda J-LDL by human fibroblasts was determined as follows. LDL with iodinated J (specific activity 200 cpm / ng) using the iodine monochloride technique of Bilheimer et al. according to J. Clin. Invest., 56, 1420-1430 (1975).

To elicit antibody-LDL interaction, 0.1 ml of each hybridoma supernatant was incubated (mixed and 125 stored) with J-LDL (final concentration 2.5 mg / ml) in 0.4 ml of Dulbecco's. minimal medium (DME) containing 2.5 mg / ml lipoprotein-free serum (LDS) for 12 hours at 4 ° C. The individually incubated mixtures were then transferred to monolayer cultures of human foreskin fibroblasts grown in DME with 10% fetal calf serum (FCS) in 10 mm diameter wells. The LDL receptors of these cells were maximally expressed upon preincubation of those cells over a period of 24 hours in DME containing 2.5 mg / ml LDS.

After individual incubations of the supernatant and fibroblast cells for 6 hours at 37 ° C, the degradation of J-LDL in the cells was determined by Drevon1 in et al. according to the method of J. Lipid res., 22, 37-46 (1981). Control cultures contained 0.4 ml of 12 5 DME containing J-LDL and one of the following: a) 0.1 ml of fresh hybridoma medium containing 20% fetal calf serum; or b) 0.1 ml of hybridoma medium from wells of hybridoma colonies that were negative for apo B-specific antibody production; c) 0.1 ml DME containing 2.5 mg / ml LDS; or d) 0.1 ml of unlabeled LDL (bringing the final concentration of unlabeled LDL in the medium to 500 ug / ml).

Il 9271 4 55 D. Isolation of anti-LDL immunoglobulin

Peritoneal fluids containing the receptors of this invention were obtained from 10-week-old Balb / c mice pretreated with 0.3 ml of mineral oil and injected intraperitoneally with 3-50 x 10 6 hybridoma cells. The average time for ascites to develop was 12 days. After clarification by centrifugation at 15,000 x g for 1 hour at 4 ° C, the peritoneal fluids were collected and stored frozen at -20 ° C.

Isolated antibody MB47 was prepared by chromatography of peritoneal fluids of a monoclonal hybridoma on a Protein A-Sepharose 4B column (Pharmacia Fine Chemicals, Piscataway, NJ). The antibody was eluted from the column with 0.1 M acetic acid.

Isolated receptors were also prepared by protein flash liquid chromatography (FPLC) on monoclonal hybridoma peritoneal fluid in a Pharmacian Mono Q HR 5/5 anion exchange column on a Pharmacian FPLC-System using a 0-0.5 M NaCl gradient in 10 mM Tris, pH 8.0, and following the instructions provided with the column.

E. Characterization of Hybridoma Antibodies. The total gamma globulin (Ig) content of each peritoneal pool was obtained by electrophoresis of 1-3 ml of samples with cellulose acetate strips in 75 mM veronal buffer, pH 8.6 for 45 minutes in a field of 200 millivolts ( mV).

The percentage of total protein that was Ig was quantified by densitometric scanning of Ponceau S-shielded gels, and total protein was determined by the modified Lowry method as previously discussed.

Mouse Ig heavy and light chains were identified by double diffusion on 0.9% agarose. Ten microliters of a suitable dilution of intraperitoneal fluid was allowed to react. equal to a large volume of appropriately diluted rabbit 56 9271 4 anti-mouse heavy and light chain specific antiserum (Litton Bionetics). After approximately 15 hours of diffusion and washing at 20 ° C, presipitine lines were identified by masking 0.5% Coomassie with brilliant blue R-250.

The isoelectric profiles of each monoclonal antibody were obtained by isoelectric focusing of 0.01 ml samples of peritoneal fluid on 0.8% agarose (EF 802-300 LKB) containing 10% sorbitol, 2% ampholine at pH 5-8 (LKB ) For 150 minutes at 3 watts constant current. After fixation and drying, the gels were stained with Coomassie brilliant blue and photographed.

1. Western blotting

Apoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of lipoproteins on a vertical slab gel strip (14x12x0.15 cm) (Hoeffer Scientific Instruments, San Francisco, CA). Gels were prepared using 25 mM Tris-glycine buffer at pH 8.6. The upper bale gel contained 1% SDS, 3% acrylamide, and the lower running gel gradient poly-acrylamide containing 1% SDS was either 3-20% or 3-6%. The lipid of the lipoproteins was removed by boiling for 3 minutes in electrophoresis sample buffer containing 1% sodium dodecyl sulfate, 10 mM Tris and 0.24 mM EDTA. Molecular weight markers and their corresponding apparent relative molecular weights were: fibrinogen, 340,000; IgG, 140,000; albumin, 69,000; ovalbumin, 43,000; soybean trypsin inhibitor, 20,500; and lysozyme, 14,300. Gels were electrophoresed for approximately 18 hours at a constant current of 13.5 milliamperes (mA).

The gels were washed in distilled water for 10 minutes and then for 10 minutes in 25 mM Tris buffer, 192 mM glycine, pH 8.3, containing 20% (v / v) methanol. Transfer to nitrocellulose (0.45 microns, Millipore Corp.) was performed by electrophoresis at 400 mA for 1 hour. The remaining active u 57 9271 4 binding sites with nitrocellulose were saturated by swimming overnight in PBS containing 3% BSA, 3% normal goat serum, 0.01% sodium azide (blocking solution).

Fixed gels or nitrocellulose transfer gels were incubated for 18 hours at 4 ° C with either immune mouse serum or peritoneal fluid appropriately diluted in PBS containing 3% BSA, 3% goat normal serum, 0.05% Tween-20 ethylene (20 monolaurate) After repeated washes, antibody binding was observed in a second 4-hour incubation at 4 ° C with 3J goat anti-immune Ig (0.5 microCi / ml) in the same buffer, followed by thorough washing.

Non-specific binding to nitrocellulose was significantly reduced by washing after incubation with both the first and second antibodies in PBS containing 3% BSA, 0.05% Tween-20 and then in 0.5 M LiCl containing 0.1% SDS. .

Gels or nitrocellulose transfer films were dried and analyzed by autoradiography (X-Omat; Eastman Kodak) at -20 ° C. If necessary, gels were stained with either 0.1% Coomassie Brilliant Blue R-250 in 50% trichloroacetic acid or silver (Bio-Rad) as described by Merril et al., Proc. Natl. Acad. Sci. USA, 7_6, 4335-4339 (1979).

F. Manufacture of Fabs

Antibody Fab fragments of isolated receptor molecules were generated by dissolving with papain. Antibody Fc portions and insoluble antibodies were removed by running on a Protein A-Sepharose-4B column. SDS-PAGE of the Fab tracks revealed two distinct bands, 25,000 and 40,000 daltons. The immunoreactivity of Fab fragments was confirmed by specific binding to LDL in a solid phase RIA (Milne et al.,

Arteriosclerosis 3, 23-30 (1983)).

58 9271 4 G. Radioimmunoassays (RIA) 125

1. Liquid phase J-labeled antigen RIA

125

To determine the fraction of J-LDL particles bound by MB47 and MB24, liquid phase RIAra / Curtiss and

Edgington, J. Biol. Chem., 25, 15213-15221 (1982). Two different LDL preparations (d = 1.019-1.063 g / ml) were studied, one isolated from the pooled plasma of 10 normal individuals and 125

the other isolated from the plasma of one normal solitary. J LDL

(2000 cpm / ng) prepared using Iodogen technology (Pierce Chemical Co., Rockford, IL) was 90% precipitated with trichloroacetic acid (TCA). It was diluted in 9% bovine serum albumin (BSA) (Sigma, St.

Louis, MO), and centrifuged at 30,000 x g for 15 minutes before each assay to remove complex material.

Assays were performed in 12x75 mm glass tubes in triplicate in 55 μM sodium barbital buffer, pH 8, containing 150 mM NaCl, 0.02% sodium azide, 3% 125. .

BSA, and 1.5 mM sodium EDTA. To 0.1 ml of J-LDL (containing 20 ng of LDL protein) was added 0.1 ml of buffer or competing antigen at increasing concentrations of 0.1 ml of isolated MB47 receptors diluted in BSA-barbital buffer. After 18 hours at 4 ° C, 0.1 mL of IgSorb (The Enzyme Co., Boston, MA) was mixed. After a maintenance time of 2 hours, 2 ml of BSA-free barbital buffer was added, and the tubes were selectively centrifuged at 1500xg for 60 minutes. The precipitates were washed twice with barbital buffer. Maximum precipitating radioactivity was determined by replacing IgSorb with 100% TCA. The minimum precipitating 125 radioactivity was determined for mucus MB47 receptors. The percent binding of J-LDL was then calculated.

125

2. Nestef donkey_J-labeled receptor RIA

Intact antibody to MB47 receptors that had been isolated. . 125 by immunopurification with iodinated J using Iodogen technique (specific activity 3000 cpm / ng) (Pierce). Thorough dialysis against PBS resulted in more than 95% of the radioactivity being precipitated 10 II; 59 9271 4

12 S

percent TCA. More than 98% of J-MB47 bound to the human LDL affinity column. The measurements were performed in triplicate in 10x75 mm silicone coated 125 .........

glass tubes. J-MB47 was added in increasing concentrations in 0.1 ml of BSA-barbital buffer to 100 ng of pooled normal human LDL diluted in 0.2 ml of BSA-barbitic buffer. Each tube contained 182 fmoles of LDL apo B (assuming a molecular weight of apo B of 550,000 daltons). After 16 hours of incubation at 4 ° C, LDL was quantitatively precipitated with lipo-protein-free rabbit antiserum specific for human LDL. (Only a density greater than 1.21 g / ml in the rabbit antiserum fraction was used because antibody MB47 binds rabbit apo B).

Preliminary studies demonstrated lipid-free rabbit antiserum. 125 concentration that precipitated 97% of 100 ng of J-human LDL. After mixing the rabbit antibody, the tubes were incubated for 16 hours at 4 ° C, and then centrifuged at 1500xg for 50 minutes at 4 ° C. The supernatants were removed and the pellets were washed twice with 2 ml of cold barbital buffer. Non-specific binding and precipitation were determined in two batches of parallel tubes.

In the first batch, no human LDL was added to the preincubation, but the same amount of rabbit second antibody was added. In another batch of tubes, non-immune rabbit serum (fraction greater than or equal to 1.21 g / ml) was replaced with immune rabbit serum antibody.

Both methods gave identical values for non-specific binding, which was linear at concentrations of 125.

growing with J-MB47 antibody, and in all cases it was less than 1% of all added activity.

125 Specific binding of J-MB47 to LDL was obtained by subtracting non-specific binding from total binding. Binding results were analyzed using a linear regression program in Scatchard analysis on ligand binding systems, βο 9271 4 which gave an estimate of antibody affinity constant (Ka) and receptor or epitope concentration (Munson et al., Anal. Biochem., 107, 220-239). ).

3. RIA Screening Assay for Solid Phase-Attached Antigen to Determine Whether Hybridoma Cultures Produce Receptors of the Invention was performed on flexible pyramid-based polyvinyl chloride microtiter plates (Dynatech, Inc., Alexandria, VA) operating in China. Lipoprotein antigens (reagent apo B-100) were fixed (bound) to the inner wall of the wells of a microtiter plate, by mixing 0.05 ml of lipoprotein in lipoprotein buffer, and incubating the plates at room temperature for 3 hours to produce a constant final bound antigen concentration and solid support. Preliminary direct binding studies using radioiodinated lipoproteins showed that there were significant differences in the efficiencies with which each of the lipoproteins bound to plastic microtiter wells. Therefore, to achieve a final bound antigen concentration of 50 ng protein / well, VLDL was used at 50 μg / ml, IDL at 6.2 μg / ml, LDL at 4.4 μg / ml, and HDL: aa 24 ug / ml. Non-specific binding sites were then blocked by mixing 0.25 ml of blocking solution into each well, incubating the mixture for one hour, and then separating the blocking solution from the wells, thus forming a solid support with low non-specific binding capacity.

From the assay, 0.050 ml of antiserum, culture supernatant, or peritoneal fluid diluted in PBS containing 3% BSA, 3% normal goat serum, and 0.05% Tween-20 / polyoxyethylene (20) sorbitan monolaurate ^ / mixed together in wells to form solid / liquid phase immunoreaction mixtures. The mixtures were then kept (allowed to immunoreact) for 18 hours at 4 ° C.

• »ll 61 9271 4

After washing the wells with PBS containing 3% BSA and 0.05% Tween-20 to remove unbound material, solid phase bound receptors were prepared for direct expression by mixing 10 ng of immunochemically purified and radioiodinated. goat anti-mouse Ig in each well to form a second solid / liquid phase mixture. This mixture was kept at 4 ° C for 4 hours to allow the labeled second receptors to bind to the first receptors bound to the solid phase and form a layered immunoreactant.

After the final wash, to remove unbound labeled receptors, individual wells were removed and counted. . . 125 _. , ... 125 _ ......,.

tnn J, with the amount of J expressed being directly proportional to the amount of first receptors bound as a solid phase immunoreactant.

The immunochemically purified second antibody was radioiodinated enzymatically using immobilized lactoperoxidase and glucose oxidase (Enzymobeads, Bio-Rad Burlingame, CA) to specific activities of 3-4 microCi / microgram.

4. Competitive RIA of the solid-phase fixed receptor _

Competitive solid phase radioimmunoassays (RIAs) for human apo Br were performed using MB47 antibody. Polyvinyl chloride wells were plated with 0.5 ml of human LDL (apo B-100 reagent) diluted in 10 μg / ml PBS, pH 7.35, and kept at 37 ° C for 2 hours. Nonspecific binding sites were blocked by baling with 5% BSA in PBS for 30 minutes at room temperature. The plates were then washed with PBS wash buffer, which additionally contained 0.1% BSA, 0.01% sodium azide, and 0.05% Tween-20.

Fresh human LDL prepared from pooled normal plasma was used as apo B-100 reagent at standard curve dilutions ranging from 0.4 μg / ml to 97.2 μg / ml. All 62 9 2 7 1 4 dilutions were made in PBS buffer containing 3% BSA, 0.01% sodium azide and 0.05% Tween-20. Standard LDL or competitors (0.025 ml) were mixed into LDL-coated wells, followed by the addition of 0.025 ml of buffer containing a solid and limiting amount of monoclonal antibody (peritoneal fluid). The optimal final antibody concentration was determined from preliminary antibody dilution studies, and the amount of antibody that results in 50% binding from the maximum was selected.

The plates were incubated for about 18 hours at 4 ° C, then washed with PBS wash buffer. Mouse antibody binding was then quantified by mixing 0.05 ml of J-immunopurified goat anti-mouse Ig (450 ng / well, 8000 cpm / ng). After a maintenance time of 4 hours at 4 ° C, the plates were washed and the counting of individual wells was performed.

H. Enzyme-linked immunosorbent assay (ELISA)

I. Non-competitive ELISA

Isolated MB47 receptors were attached to the walls of polystyrene microtiter plate wells (Nunc-Immuno Plate I) by mixing 0.15 ml of sodium bicarbonate buffer pH 9.0 containing 1 ug / ml receptor protein in each well. The wells were incubated for 16 hours at 4 ° C, and then washed 3 times with PBS containing 0.1% BSA and 0.05% Tween. The remaining non-specific binding sites were then blocked by mixing in each well.

0.2 ml BSA in PBS, incubating the mixture for 1 hour at 23 ° C, and washing thereafter as described above. Thus, pre-prepared wells (solid carriers) can be used for up to about one month after preparation when stored in a humidified chamber.

Apo B-100 reagent (human LDL) was diluted in PBS to concentrations ranging from 2.0 to 0.062 ug / ml used. as standard control solutions. Plasma samples were diluted 1: 2000 in PBS.

II: '92714

Fifty microliters of standard or sample was mixed into the wells in triplicate. Within approximately one minute of Vilde, 50 ul of PBS containing mg / ml HRPO-labeled MB24 receptors was mixed in each well. Immunoreaction mixtures were maintained at 25 ° C for 30 minutes. The unbound material was then separated from the wells by washing as described above.

The amount of layered immunoreactant attached to the solid phase containing the HRPO label was then determined by mixing 0.1 ml of freshly prepared substrate solution (distilled water containing 3% H 2 O 2 and 0.67 mg / ml o-phenylenediamine ( OPD)) to each well. The varin was allowed to develop for 30 minutes at 25 ° C. The substrate conversion reaction was then stopped by stirring 0.05 ml of 4N H 2 O 2 in each well. The optical densities (O.D.) of the solutions were determined at a wavelength of 490 nanometers (nm) using a Dynatech MR600 (Dynatech, Alexandria, VA) microtiter plate reader.

2. Competitive ELISA

Apo B-100 reagent was attached to the well walls of a flexible polyvinyl chloride microtiter plate (Microtest III,

Falcon Labware, Becton, Dickinson & Co., Oxnard, CA) as a solid matrix, by mixing 0.2 ml of PBS containing 5 μg / ml isolated human LDL in each well. The wells were kept for 16 hours at 4 ° C and then washed 3 times with 0.2 ml of PBS containing 1% BSA, 0.5% Tween and 0.02% aprotinin (Sigma Chemical Co.). . Non-specifically binding sites shared on the foot were blocked as described in the non-competitive ELISA.

For the standard curve included in each plate, apo B-100 reagent was diluted in PBS containing 0.5% lipoprotein-free plasma (LPDP) to give concentrations ranging from 32 mg / ml to 0.25 mg / ml.

9271 4 64

Plasma samples were diluted 1: 200 in PBS containing 0.5% LPDP. Fifty microliters of standards or samples were mixed in triplicate in the wells. Within about 5 minutes thereafter, 50 μl of PBS containing 3% BSA and about 4 μg / ml MB24 receptors were mixed in each well. The mixtures thus formed were kept at 4 ° C for about 18 hours. The unbound material was then separated from the solid phase MB24 reagent apo B-100 immunoreaction products by washing as described above.

Solid phase immunoreactants were prepared for the assay by mixing 0.1 ml of PBS containing 1% BSA and an effective amount of HRPO-labeled goat anti-mouse IgG in each well. This second immunoreaction mixture was kept at 24 ° C for about 1 hour, and then washed as described above to form a layered immunoreactant.

The amount of solid phase attached layered immunoreactant containing HRPO label was determined as described in the competitive ELISA.

I. Plasma Samples and Lipoprotein Quantification Plasma samples were obtained from 20 patients with coronary artery disease from a cardiac catheter laboratory in San Diego, VA Hospital, and 20 patients with normal hypercholesterolemia from the University of California, San Diego Clinic. In addition, plasma was obtained from 20 normal individuals.

Blood was collected into tubes containing 1.5 mg / ml ethylenediaminetetraacetate (EDTA), and plasma was immediately separated by centrifugation at 4 ° C.

Plasma total cholesterol and triglycerides were measured from fresh plasma samples in a standardized clinical laboratory using an Abbot ABA-200 dichromate analyzer, and Boehringer-Mannheim High Power Cholesterol Reagent 236691 and Abbot Laboratories Triglycerides A-Gent. LDL

II

92714 6 5 and HDL cholesterol were measured using the technique described in Lipid Research Clinic Procedures, HEW Pub. Well. 75-628 (NIH), 2 pp., Washington, D.C., Gov. Vistaprint. Off., (1974). Apoprotein B levels were determined using two commercially available radiant immunodiffusion kits: Diffen RID (Tago, Inc., Burlingame, CA), designated RID # 1, and M-Partigen RID, (Calbiochem-Behring, La Jolla, CA). , designated RID # 2 in this.

J. Binding of LDL to MB47-Sepharose-4B Column 50 milligrams of substantially pure MB47 receptors, obtained by protein A column chromatography, were coupled to 12 ml of cyanogen bromide-activated Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, NY) according to the manufacturer's instructions. -sesti. Subsequently, 10 mg of LDL was added to the column and incubated overnight (about 16 hours) at 4 ° C. Unbound LDL was then eluted and assayed for LDL protein as previously described.

The foregoing patent application, including specific embodiments, is intended to illustrate the present invention and should not be construed as limiting. Numerous other variations and modifications may be made without departing from the true spirit and scope of the novel compositions of the invention.

Claims (19)

1. Hybridoma with ATCC deposit number HB 8746. 2. Receptor, characterized in that it (a) immunoreacts with apoprotein B-100, and (b) is secreted by hybridomas with ATCC deposit number HB 8746. 3. Receptor according to claim 2, characterized by having an intact antibody. 4. Hybridoma with ATCC deposit number HB 8742. 5. Receptor, characterized in that it a) immunoreacts with apoprotein B-100, and (b) is secreted by hybridomas with ATCC deposit number HB 8742. 6. Receptor according to claim 5, characterized in that it has an intact antibody. A method for determining the amount of apoprotein B-100 in a body fluid sample, characterized in that the method comprises the following steps: (a) the body fluid sample to be determined is taken; (b) receptor molecules are produced in biologically active form which (i) immunoreact with apoprotein B-100 and (ii) secreted by hybridomas with ATCC deposit number HB 8746 or hybridomas with ATCC deposit number HB 8742; (c) mixing a plurality of said body growth sample with a predetermined amount of said receptor molecule to form an immunoreaction mixture; (d) said mixture is maintained in biological assay conditions for a predetermined time which, with respect to the receptor molecules in the mixture, is sufficient to immunologically bind apoprotein B-100 present in the sample, thereby forming immunoreactant; and (e) the amount of immunoreactant formed in said mixture is constituted. 92714 Method according to claim 7, characterized in that said body fluid sample is either plasma or serum. The method of claim 7, characterized in that said receptor is an intact antibody. A method according to claim 7, characterized in that the body fluid sample is further prepared for the determination by including five additional steps: (f) producing biologically active other receptor molecules which immunoreact with apoprotein B-100 contained in said body fluid sample, and which residues of each receptor molecule set secreted by hybridoma with ATCC deposit number HB 8746 or hybridoma with ATCC deposit number HB 8742; and (g) mixing a predetermined amount of said second receptor molecule with said body fluid sample, forming an immunoreaction mixture; (h) said immunoreaction mixture is maintained in biological conditions for a predetermined time which, with respect to the receptor molecules in the mixture, is sufficient to immunologically bind apoprotein B-100 present in the sample to form stored immunoreactant. The method of claim 10, characterized in that said receptor is an intact antibody. Method according to claim 10, characterized in that the aforementioned receptor is fixed to a solid matrix to form a solid support; said second receptor contains a plunger with the help of spring the presence of said second receptor contained in the immunoreactant may be expressed; and the immunoreactant determined in step (e) is a solid phase stored immunoreactant containing said plunger. A diagnostic system for determining the amount of apo-B-100. a body sample, characterized in that it comprises: (a) a first biologically active, specifically bound subject comprising receptor molecules which (i) immunoreact with apoprotein B-100, and (ii) are either receptor molecules secreted by ATCC HB 8746 hydride or receptor molecules secreted by ATCC HB 8742? and (b) a biologically active, platelet, second, specifically bound, subject to express the immunoreaction in which said understood bound subject participates with apo B-100. 14. Determination system according to claim 13, characterized in that the stamped, second specifically bound substance comprises other receptor molecules either of receptors secreted by hybridoma ATCC HB 8746 or receptors secreted by hybridoma ATCC HB 8742. 15. Determination system according to claim 14, characterized in that the plunger is an enzyme. 16. Determination system according to claim 13, characterized in that said first specifically bound subject is obtained on a fixed matrix, whereby a fixed carrier is formed. 17. Sterile affinity sorbent formed by biologically active receptor molecules that immunoreact with apoprotein B-100 and are selected in the group consisting of:. (a) receptor molecules secreted by hybridoma HB 8746 (b) receptor molecules secreted by hybridoma HB 8742 and (c) a mixture of receptor molecules under points (a) and (b); characterized by the fact that said receptor molecules were fixed to a solid matrix, whereby a solid carrier is formed. «♦ • A method for determining apoprotein B-100 contained in a body fluid sample, characterized in that it comprises the sixth step: (a) the body fluid sample to be determined; (b) producing a solid carrier formed from a solid matrix, in which is additionally obtained in biologically active form 92714 a first receptor that reacts immunologically with apoprotein B-100 and which is secreted by hybridomas with ATCC deposit number HB 8746; (c) a second biologically active receptor is produced which immunoreacts with apoprotein B-100 and is secreted by hybridomas with ATCC deposit number HB 8742, while said second receptor is associated with a stem enzyme of ours in the presence of said second receptor. expressed in the immunoreactant; (d) substantially simultaneously mixed: (i) said body sample; and (ii) said solid carriers; and (iii) said stamped second receptor to form a solid / liquid phase immunoreaction mixture; (e) said mixture is maintained in biological assay conditions for a predetermined period which, with respect to said understanding receptor and said second stamped receptor, is sufficient to immunologically bind apoprotein B-100 present in the sample, wherein a stored immunoreactant in solid phase and liquid phase are formed; (f) separating said solid phase immunoreactant from said liquid phase; and (g) the plurality of stamped second receptor is constituted which is bound to the solid phase stored immunoreactant. A competitive method for determining apoprotein B-100 in a body fluid sample, characterized in that it comprises the sixth step: (a) the body fluid sample to be determined is taken; (b) producing a solid support comprising a solid matrix, in addition to which a predetermined amount of reagent apoprotein B-100 is obtained; (c) substantially simultaneously mixed: (i) said body sample; (ii) said solid carrier, (iii) a predetermined amount of receptor molecules immunoreacting with apoprotein B-100, either excreted from * hybridomas with ATCC deposit number HB 8746 or from hybrid judgment with ATCC deposit number HB 8742, forming a solid / liquid phase mixture; (d) said mixture is maintained in biologically determined assay conditions for a period which, with respect to receptor molecules, is sufficient to immunologically bind to apoprotein B-100 molecules in a solid carrier and apoprotein B-100 molecules in the body fluid sample and to form a solid phase immunoreactant and a liquid phase immunoreactant; and (e) said solid phase immunoreactant is separated from said liquid phase; (f) by adding in said solid phase immunoreactant a biologically active second receptor, a mixture is reacted which second receptor reacts with said understood receptor, forming a second solid / liquid phase immunoreaction mixture in which said second receptor coupled to the enzyme stamp is allowed to expressing the presence of said second receptor in immunoreactant one; (g) maintaining said second mixture for a time which, with respect to the stamped second receptor, is sufficient to immunologically bind the entire fastest receptor present as a solid immunoreactant, forming a solid phase immunoreactant; and (h) separating said stored solid phase immunoreactant from said liquid phase; and (i) determining the amount of stamped second receptor, which is bound to said solid phase stored immunoreactant.